Compounds for treating mitochondrial disease

ABSTRACT

The invention relates to novel compounds that are useful for modulating cellular ROS. The compounds are amide-derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoic acid. The compounds of the invention are formulated into pharmaceutical or cosmetic compositions. The invention further relates to methods wherein the compounds of the invention are used for treating or preventing diseases associated with increased ROS levels mitochondrial disorders and/or conditions associated with mitochondrial dysfunction, including adverse drug effects. The invention also relates to cosmetic methods for treating or delaying further aging of the skin and veterinary applications.

FIELD OF THE INVENTION

The invention relates to the field of human and animal diseases andcosmetics. The invention in particular relates to amide-derivative of2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoicacid for treating conditions that are associated with oxidative stress,mitochondrial dysfunction or mitochondrial deficiencies, includingadverse drug effects causing oxidative stress or mitochondrialdysfunction, and for cosmetic use against aging of the skin.

BACKGROUND OF THE INVENTION

Reactive oxygen species (ROS) are involved in a broad spectrum ofcellular processes, such as cell signalling, apoptosis and homeostasis.This implicates a crucial role for ROS in normal cellular function.However, (too) high levels of ROS may cause significant damage to cellstructures, which is known as oxidative stress. Oxidative stress isthought to be involved in the development of many different diseases,such as Asperger syndrome, ADHD, cancer, Parkinson's disease, Laforadisease, Alzheimer's disease, atherosclerosis, heart failure, myocardialinfarction, fragile X syndrome, Sickle Cell Disease, lichen planus,vitiligo, autism, infection, congenital muscular dystrophies,dystrophinopathies and chronic fatigue syndrome. Furthermore, it hasrecently been shown that supporting the cellular redox homeostasis isalso important in many ex vivo techniques, such as during the inductionof pluripotent stem cells (Ji et al, Stem cell reports (2014) vol. 2:44-51).

A major source of reactive oxygen species is mitochondria. Mitochondriaare essential organelles that constitute the ‘powerhouses’ of the cell.Defects in these organelles often lead to a variety of severe metabolicdisorders affecting the organs that have a high-energy demand, such asmuscle and brain. With an incidence of at least 1 in 5000 individuals itis recognized as the most common group of inborn errors of metabolism.Moreover, because programmed cell death (apoptosis) is triggered bymitochondria, defects in these organelles have consequences far beyondthe diseases, which brought them initially to our attention andinvolvement in cancer and neurodegenerative diseases like Alzheimer andParkinson has been demonstrated. Many commonly used drugs like theNRTIs, certain antibiotics and anti-epileptic drugs, may causemitochondrial dysfunction. So far no effective treatment is available tocure or improve these disease conditions.

One of the primary functions of mitochondria is oxidativephosphorylation (OXPHOS). The molecule adenosine triphosphate (ATP)functions as an energy “currency” or energy carrier in the cell, andeukaryotic cells derive the majority of their ATP from biochemicalprocesses carried out by mitochondria, including the citric acid cycle,which generates reduced NADH+H⁺ from oxidized NAD⁺, and OXPHOS, duringwhich NADH+H⁺ is oxidized back to NAD⁺. The electrons released byoxidation of NADH+H⁺ are shuttled down a series of protein complexes(Complex I, Complex II, Complex III, and Complex IV) known as themitochondrial respiratory chain. These complexes are embedded in theinner membrane of the mitochondrion. Complex IV, at the end of thechain, transfers the electrons to oxygen, which is reduced to water. Theenergy released as these electrons traverse the complexes is used togenerate a proton gradient across the inner membrane of themitochondrion, which creates an electrochemical potential across theinner membrane. Another protein complex, Complex V (which is notdirectly associated with Complexes I, II, III and IV) uses the energystored in the electrochemical gradient to convert ADP into ATP.

Mitochondrial oxidative phosphorylation is a major cellular source ofreactive oxygen species (ROS), as approximately 1-2% of oxygen consumedduring physiological respiration is converted into superoxide (.O₂ ⁻)when electrons prematurely leak from the electron transport chain andare aberrantly transferred to molecular oxygen. However, under specificmetabolic or stress conditions, more electrons can prematurely exit therespiratory chain to further augment mitochondrial superoxidegeneration. Leakage occurs at complex I, complex II or complex III,although complex I and complex III are the major sites of superoxidegeneration within mitochondria (Philip West A, Nature Reviews Immunology2011 (11):389-402).

The contribution of mitochondrial dysfunction to human disease wasalready recognised in the late 1980s, when maternally inherited pointmutations, as well as deletions arising spontaneously duringdevelopment, were found to be associated with rare neurologicalsyndromes. Mitochondrial dysfunction contributes to various diseasestates. Some mitochondrial diseases are due to mutations or deletions inthe mitochondrial genome. If a threshold proportion of mitochondria inthe cell is defective, and if a threshold proportion of such cellswithin a tissue have defective mitochondria, symptoms of tissue or organdysfunction can result. Practically any tissue can be affected, and alarge variety of symptoms may be present, depending on the extent towhich different tissues are involved. Some examples of mitochondrialdiseases are Friedreich's ataxia (FRDA), Leber's Hereditary OpticNeuropathy (LHON), dominant optic atrophy (DOA); mitochondrial myopathy,encephalopathy, lactacidosis, and stroke (MELAS), Myoclonus EpilepsyAssociated with Ragged-Red Fibers (MERRF) syndrome, Leigh syndrome, andoxidative phosphorylation disorders. Mitochondrial diseases involvechildren and adults who manifest the signs and symptoms of acceleratedaging, including neurodegenerative diseases, stroke, blindness, hearingimpairment, diabetes, and heart, liver and kidney failure andmyopathies.

Very few treatments are available for patients suffering from thesemitochondrial diseases. The drug idebenone (a CoQ₁₀ variant) has beenapproved for the treatment of Friedreich's ataxia (Benit et al., 2010,Trends Mol Med, 16:210-7; Klopstock et al., 2011, Brain, 134:2677-86)and LHON. Another compound, MitoQ₁₀ (mitoquinone), has been proposed fortreating mitochondrial disorders (U.S. Pat. No. 7,179,928) but clinicalresults for MitoQ have not yet been reported. A successful treatmentstrategy has been developed for patients with a secondary mitochondrialdisorder involving Ullrich's congenital muscular dystrophy and Bethlem'smyopathy. The pathogenic mechanism in these myopathies involvesinappropriate opening of the mitochondrial permeability transition pore.This action was prevented in patients treated with thepermeability-transition-pore desensitizer CSA (cyclosporin A; Angelin etal., 2007, Proc Natl Acad Sci USA, 104:991-6; Merlini et al., 2008, ProcNatl Acad Sci USA, 105:5225-9).

An overview of current clinical trials relating to mitochondrial diseasecan be found online(www.clinicaltrials.gov/ct2/results?term=mitochondrial+disease); thisinclude studies of CoQ10 for the treatment of muscle weakness andmitochondrial diseases, dietary supplements for MELAS, EPI-743 formitochondrial diseases, human growth hormone for obesity, nutritionaltherapy for diabetes, pioglitazone for diabetes, idebenone for MELAS,and vitamin E for mitochondrial trifunctional protein deficiency.

WO 2012/019032 discloses methods of treatment, prevention, orsuppression of symptoms associated with a mitochondrial disorder and/ormodulating, normalizing, or enhancing one or more energy biomarkers,whereby vitamin K analogues are administered.

WO 2012/019029 discloses methods of treatment, prevention, orsuppression of symptoms associated with a mitochondrial disorder and/ormodulating, normalizing, or enhancing one or more energy biomarkers,whereby naphtoquinones and derivatives thereof are administered.

Distelmaier et al. (2012, Antioxid Redox Signal. 17 (12):1657-69)disclose that Trolox™ (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid) reduces the levels of ROS, increased mitofusins-mediatedmitochondrial filamentation and expression of mitochondrial complex I,activity of citrate synthase and OXPHOS enzymes and cellular 02consumption in cultured healthy human skin fibroblasts.

WO 2014/011047 discloses methods for treating or preventingmitochondrial disorders, conditions associated with mitochondrialdysfunction and/or neoplastic diseases, using Trolox-derivatives. Inparticular, these derivatives can be used in modulating mitochondrialmorphology and/or expression of OXPHOS enzymes and/or cellular ROS. WO2014/011047 discloses Trolox derivatives, wherein the carboxylic acidmoiety is replaced by an amide moiety and wherein the nitrogen atom ofthe amide moiety is connected via a linker to a cationic nitrogen atom.

Besides the role of ROS in a wide variety of diseases and conditions,excessive ROS levels also play an important role during thetransformation of somatic cells into induced pluripotent stem cells. Inparticular, increased levels of ROS and oxidative stress damage has beenobserved during the early stages of reprogramming somatic cells intopluripotent stem cells. Notably, the addition of antioxidants appearedto reduce both ROS and double stranded breaks, resulting in lowerapoptotic rates (Ji et al, supra). There is however still a need in theart for effective means to modulate ROS levels. Furthermore, there isstill a need for effective means for modulating mitochondrial functionfor them to be used in treatments of mitochondrial disease and/orconditions associated with mitochondrial dysfunction or for cosmeticuse.

SUMMARY OF THE INVENTION

In a first aspect the invention pertains to novel amide-derivatives of2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoicacid, which are represented by general structure (I):

Herein,

-   -   L is a linker between the amide nitrogen atom and the distal        nitrogen atom (N*) comprising 1 to 10 optionally substituted        backbone atoms selected from carbon, nitrogen and oxygen;    -   R¹ and R² are each independently selected from hydrogen (H),        C₁-C₆ alkyl or C₁-C₆ alkenyl, or R¹ and R² are joined together        and thus form a second linker between the amide nitrogen atom        and the distal nitrogen atom, or R¹ is joined with a backbone        atom of the linker L in a cyclic structure and/or R² is joined        with a backbone atom of the linker L in a cyclic structure;    -   R³ is selected from hydrogen (H), C₁-C₆ alkyl or C₁-C₆ alkenyl,        wherein the alkyl or alkenyl moiety may be substituted with one        or more halogen atoms or (halo)alkoxy moieties, or R³ is absent        when the distal nitrogen atom is part of an imine moiety; and    -   in case the distal nitrogen atom is in cationic form (i.e. the        compound is in salt form): R⁴ is selected from hydrogen (H) or        C₁-C₆ alkyl, wherein the alkyl moiety may be substituted with        one or more halogen atoms or (halo)alkoxy moieties and X is an        anion, preferably a pharmaceutically acceptable anion; or    -   in case the distal nitrogen atom is in neutral form: R⁴ and X        are absent.

Preferred compounds according to the first aspect of the invention arethose wherein R⁴═H and X═Cl⁻ or wherein R⁴ and X are absent and wherein:

-   -   L=L, R¹-R²=L¹, R³═H;    -   L=L¹, R¹═H, R²═H, R³═H;    -   L=L², R¹═H, R²═H, R³═H;    -   L=L³, R¹═H, R²═H, R³═H;    -   L=L⁴, R¹═H, R²═H, R³═absent;    -   L=L⁵, R¹═H, R²═H, R³═absent;    -   L=L⁶, R¹═H, R²═H, R³═absent;    -   L=L³, R¹═H, R²=Me, R³=Me;    -   L=L¹, R¹═H, R²=Me, R³=Me;    -   L=L⁷, R¹═H, R²═H, R³═absent;    -   L=L¹, R¹═H, R²═H, R³═absent;    -   L=L⁹, R¹═H, R²═H, R³═absent;    -   L=L¹⁰, R¹-R^(1′)=L¹, R²═H, R³═absent;    -   L=L¹¹, R¹═H, R²═H, R³═H;    -   L=L¹², R¹═H, R²═H, R³═absent;    -   L=L¹³, R¹═H, R²═H, R³═H;    -   L=L¹⁴, R¹═H, R²═H, R³═H;    -   L=L¹⁵, R¹═H, R²═H, R³═H;    -   L=L¹⁵, R¹═H, R²=Me, R³=Me    -   L=L¹⁶, R¹═H, R²═H, R³═H;    -   L=L¹⁷, R¹═H, R²═H, R³═H;    -   L=L¹⁶, R¹═H, R²=Me, R³=Me;    -   L=L¹⁸, R¹═H, R²-R^(2′)=L³, R³═H;    -   L=L¹⁹, R¹═H, R²-R^(2′)=L, R³═H;    -   L=L²⁰, R¹═H, R²═H, R⁵-R^(5′)=L³, R³═absent;    -   L=L²¹, R¹═H, R²-R^(2′)=L¹, R³═H;    -   L=L²², R¹-R^(1′)=L¹, R²=TH, R³═H;    -   L=L²³, R¹-R^(1′)=L¹, R²=TH, R³═H;    -   L=L²⁴, R¹-R^(1′)=L³, R²═H, R³═H;    -   L=L²⁵, R¹-R^(1′)=L³, R²═H, R³═absent;    -   L=L²⁶, R¹═H, R²═H, R⁵-R^(5′)=L¹, R³═H.    -   L=L¹⁹, R¹═H, R²-R^(2′)=L³, R³=Me;    -   L=L¹⁹, R¹═H, R²-R^(2′)=L¹, R³═H; or    -   L=L²¹, R¹═H, R²-R^(2′)=L¹, R³=Me.

In a second aspect, the invention relates to a compound according to theinvention for use as a medicament.

In a further aspect, the invention pertains to method of treating,preventing, or suppressing symptoms associated with a pathology,condition or disorder associated with an increased or a decreased ROSlevel, the method comprising administering to a subject an effectiveamount of one or more compounds according to the invention as definedherein above. Alternatively, the invention relates to a compoundaccording to the invention for use in treating, preventing orsuppressing symptoms associated with a pathology, condition or disorderassociated with an increased or a decreased ROS level. Preferably, thecompound according to the invention can be used in treating, preventingor suppressing symptoms associated with a pathology, condition ordisorder associated with an increased ROS level.

In a further aspect, the invention relates to a method of treating,preventing, or suppressing symptoms associated with a mitochondrialdisorder or with a condition associated with mitochondrial dysfunction,the method comprising administering to a subject an effective amount ofone or more compounds according to the invention as herein definedabove. Alternatively, the invention relates to a compound according tothe invention for use in a method of treating, preventing, orsuppressing symptoms associated with a mitochondrial disorder or with acondition associated with mitochondrial dysfunction.

Further aspects of the invention relate to the cosmetic use of thecompounds according to the invention, wherein the compounds may be used(in methods) to revive the skin of a treated individual, particularly inindividuals with aged skin, either due to aging or due to excessiveexposure to sun, to compounds according to the invention for use as aconservative agent, for use in research applications, such as in vitro,in vivo, or ex vivo experiments in order to modulate one or more energybiomarkers in an experimental system, for use in biochemical tests orassays.

In yet further aspects, the invention pertains to pharmaceutical orcosmetic composition comprising a compound according to the invention,and to articles of manufacture and kits comprising a compound accordingto the invention. A particular aspect of the invention concerns acosmetic method for treating or delaying further aging of the skin in asubject, the method comprising the step of administering to the skin ofthe subject an effective amount of a composition comprising a compoundas defined herein. The compounds according to the invention aresurprisingly effective in reducing ROS levels, as evidenced in theexamples, and are thus particularly suitable in such a method.

In another aspect, the invention relates to an ex vivo method forscavenging reactive oxygen species in a cell, wherein the methodcomprises a step of exposing the cell to a compound of the invention.The compounds according to the invention are surprisingly effective inreducing ROS levels, as evidenced in the examples, and are thusparticularly suitable in such a method. Preferably, the cell is asomatic cell, a gamete, a gametocyte or an undifferentiated stem cell.More preferably, the somatic cell is further reprogrammed to an inducedpluripotent stem cell (iPSC) by exposing the cell to at least onereprogramming factor. The reprogramming factor is preferably at leastone of Oct4, Sox2, KLF4, c-Myc, Lin28, Nanog, Glis1, Sall4, Esrrb andNr5a2 and the somatic cell is preferably a fibroblast, neuronal(progenitor) cell, hepatocyte, B cell, kidney cell, muscle cell, adrenalgland cell, keratinocyte, melanocyte, epithelial cell or a peripheralblood derived cell, preferably wherein the peripheral blood derived cellis an endothelial progenitor cell (L-EPCs) or a cord blood derived celltype (CD34+).

DESCRIPTION OF THE INVENTION

In a first aspect the invention pertains to a compound which is anamide-derivative of2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-butanoicacid (compound A). Compound A is the ring-opened form of6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Compound B),which is also known under trademark name Trolox. Compound A is alsoreferred to as “open-Trolox”.

In a compound of the invention, the carboxylic acid moiety of compound Ais replaced by an amide moiety, wherein the nitrogen atom of the amidemoiety is connected via a linker to a distal nitrogen atom. In thecontext of the present invention, the nitrogen atom that is closest tothe open-Trolox core of compound A is referred to as the “amide nitrogenatom” and the nitrogen atom that is farthest away from the open-Troloxcore is referred to as the “distal nitrogen atom”. Additional nitrogenatoms may be present in the linker. The distal nitrogen atom may be inneutral form or in cationic form.

The compound of the invention may be identified by general structure(I):

Herein,

-   -   L is a linker between the amide nitrogen atom and the distal        nitrogen atom (N*) comprising 1 to 10 optionally substituted        backbone atoms selected from carbon, nitrogen and oxygen;    -   R¹ and R² are each independently selected from hydrogen (H),        C₁-C₆ alkyl or C₁-C₆ alkenyl, or R¹ and R² are joined together        and thus form a second linker between the amide nitrogen atom        and the distal nitrogen atom, or R¹ is joined with a backbone        atom of the linker L in a cyclic structure and/or R² is joined        with a backbone atom of the linker L in a cyclic structure;    -   R³ is selected from hydrogen (H), C₁-C₆ alkyl or C₁-C₆ alkenyl,        wherein the alkyl or alkenyl moiety may be substituted with one        or more halogen atoms or (halo)alkoxy moieties, or R³ is absent        when the distal nitrogen atom is part of an imine moiety; and    -   in case the distal nitrogen atom is in cationic form (i.e. the        compound is in salt form): R⁴ is selected from hydrogen (H) or        C₁-C₆ alkyl, wherein the alkyl moiety may be substituted with        one or more halogen atoms or (halo)alkoxy moieties and X is an        anion, preferably a pharmaceutically acceptable anion; or    -   in case the distal nitrogen atom is in neutral form: R⁴ and X        are absent.

To distinguish between the amide nitrogen atom and the distal nitrogenatom, the latter one is labelled N* in general structure (I). Thecompound identified by general structure (I) comprises at least onechiral carbon atom (stereocenter), i.e. the atom at the 2-position ofthe butanoic acid-moiety. Both the compound having an S-configuration asthe compound having an R-configuration of the carbon atom at the2-position are encompassed in the present invention, as well as mixturesof the different stereoisomers. Such a mixture may have one of theconfigurations in enantiomeric excess, or may be racemic. Whenever oneor more additional stereocenters are present in the compound accordingto the invention, for example in linker L, each may individually existin the S-configuration, in the R-configuration, or as a mixture of bothconfigurations. Such a mixture may have one of the configurations inenantiomeric excess, or may be racemic. In case addition stereocentersare present, all diastereomers of the compound of general structure (I),in each possible ratio, are encompassed in the present invention.

In a preferred embodiment, the solubility of the compound of theinvention in water, expressed as log(P_(ow)) is between 2.0 and 5.0,preferably between 2.5 and 4.5, more preferably between 3.0 and 4.0.Log(P_(ow)), the logarithm of the partition coefficient between1-octanol and water, is a well-known measure of water solubility.Compounds having a log(P_(ow)) value between 3 and 4 are ideallybalanced between sufficient water solubility for preparation of aqueoussolutions or suspensions and sufficient lipophilicity to ensureefficient transport of the compound over the cellular membrane. Theskilled person will appreciate how to determine which combinations of L,R¹, R², R³, R⁴ and X as defined herein to afford a compound having alog(P_(ow)) value between 3 and 4. Suitable tests to define thelog(P_(ow)) value of a compound are well-known to the skilled person,and include but are not limited to the shake-flask method, ITIES, thedroplet method or using HPLC. The log(P_(ow)) of a compound can also bepredicted using QSPR algorithms.

R¹ and R² are each independently selected from hydrogen (H), C₁-C₆ alkylor C₁-C₆ alkenyl, or one or both of R¹ and R² are embedded in a cyclicstructure as described here below. Preferably, R¹ is H or C₁-C₂ alkyl orR¹ and R² are joined together and thus form a second linker between theamide nitrogen atom and the distal nitrogen atom, or R¹ is joined with abackbone atom of the linker L in a cyclic structure, more preferably R¹is H or C₁-C₂ alkyl, even more preferably R¹ is H or methyl (Me), mostpreferably R¹ is H. Preferably, R² is H or C₁-C₂ alkyl or R¹ and R² arejoined together and thus form a second linker between the amide nitrogenatom and the distal nitrogen atom, or R² is joined with a backbone atomof the linker L in a cyclic structure, more preferably R² is H or C₁-C₂alkyl, even more preferably R² is H or methyl (Me), most preferably R²is H. In an especially preferred embodiment, R² is joined with abackbone atom of the linker L in a saturated cyclic structure, asfurther defined below, preferably a piperidine ring.

In one embodiment, the amide nitrogen atom is connected to the distalnitrogen atom via a second linker. This second linker is defined byjoining together R¹ on the amide nitrogen atom and R² on the distalnitrogen atom. Thus, the amide nitrogen atom, the distal nitrogen atom,the first linker and the second linker together form a cyclic structure,preferably a 4-10-membered cyclic structure, more preferably a5-8-membered cyclic structure, most preferably a 6-membered cyclicstructure. In a preferred embodiment, the second linker is a —CH₂—CH₂—or —CH₂—CH₂—CH₂— bridge, most preferably a —CH₂—CH₂— bridge, wherein twoor three, preferably two, carbon atoms are present between the amidenitrogen atom and the distal nitrogen atom.

In another embodiment, the amide nitrogen atom is connected to abackbone atom of the linker via a second linker, thereby forming acyclic structure, preferably a 4-10-membered cyclic structure, morepreferably a 5-8-membered cyclic structure, most preferably a 6-memberedcyclic structure. The backbone atom of the linker to which the nitrogenatom is connected in this respect has a substituent R^(1′), which isjoined together with R¹ on the amide nitrogen atom. Thus, the amidenitrogen atom, part of first linker located between the amide nitrogenatom and the atom bearing R^(1′), the backbone atom bearing R^(1′) andthe second linker together form the cyclic structure. In thisembodiment, the distal nitrogen atom is not included in this cyclicstructure, but instead only part of the backbone of the linker isincluded. In a preferred embodiment, this connection between the amidenitrogen atom and a backbone atom of the linker is a —CH₂—CH₂— or—CH₂—CH₂—CH₂— bridge, most preferably a —CH₂—CH₂— bridge, wherein two orthree, preferably two, carbon atoms are present between the amidenitrogen atom and the backbone atom of the linker. Most preferably, thecyclic structure containing the amide nitrogen atom is a fully saturatedring, preferably selected from a piperidine ring, a pyrrolidine ring, apiperazine ring, an imidazolidine ring, a pyrazolidine ring and anazepane ring, more preferably a piperidine ring or a pyrrolidine ring,most preferably a piperidine ring.

In another embodiment, the distal nitrogen atom is connected to abackbone atom of the linker via a second linker, thereby forming acyclic structure, preferably a 4-10-membered cyclic structure, morepreferably a 5-8-membered cyclic structure, most preferably a 6-memberedcyclic structure. The backbone atom of the linker to which the nitrogenatom is connected in this respect has a substituent R^(2′), which isjoined together with R² on the distal nitrogen atom. Thus, the distalnitrogen atom, part of first linker located between the distal nitrogenatom and the atom bearing R^(2′), the backbone atom bearing R^(2′) andthe second linker together form the cyclic structure. In thisembodiment, the amide nitrogen atom is not included in this cyclicstructure, but instead only part of the backbone of the linker isincluded. In a preferred embodiment, this connection between the distalnitrogen atom and a backbone atom of the linker is a —CH₂—CH₂— or—CH₂—CH₂—CH₂— bridge, most preferably a —CH₂—CH₂— bridge, wherein two orthree, preferably two, carbon atoms are present between the distalnitrogen atom and the backbone atom of the linker. Most preferably, thecyclic structure containing the distal nitrogen atom is a fullysaturated ring, preferably selected from a piperidine ring, apyrrolidine ring, a piperazine ring, an imidazolidine ring, apyrazolidine ring and an azepane ring, more preferably a piperidine ringor a pyrrolidine ring, most preferably a piperidine ring. It is alsopossible that a connection exists between R¹ on the amide nitrogen atomand an R^(1′) substituent on the linker and between R² on the distalnitrogen atom and an R^(2′) substituent on the linker.

Among the above-mentioned possibilities for R², it is most preferredthat the distal nitrogen atom is connected to a backbone atom of thelinker via a second linker wherein R² is joined with R^(2′), as furtherdefined here above. Among the tested compounds, these have been foundmost active.

When the distal nitrogen atom is part of an imine moiety, the linker Lcomprises at least one double bond located between the distal nitrogenatom and the adjacent backbone atom of the linker, or R² comprises atleast one double bond located between the distal nitrogen atom and theadjacent atom of R² (i.e. R²═C₁-C₆ alkenyl). In such instances, R³ isabsent. In the context of the present invention, the distal nitrogenbeing part of an imine moiety includes instances wherein the distalnitrogen atom is part of an heteroaromatic ring, in particular a pyrrolering, a pyridine ring or a imidazole ring, in which instances a doublebond is formally present between the distal nitrogen atom and theadjacent carbon atom either in the linker or in R². Preferred moietiescomprising an imine moiety include guanidine, amidine and pyridine. Forguanidine and amidine, one of the nitrogen atoms is substituted to formthe connection with the amide nitrogen atom via linker L. For pyridine,one of the carbon atoms is substituted. When the distal nitrogen atom ispart of an amine moiety, it is connected to the linker and R² via twosingle bonds, and R³ is present. It is preferred that the distalnitrogen atom is part of an amine moiety, i.e. having three or foursingle bonds to each of R¹, R², R³ and optionally R⁴.

In the instance that R³ is present, R³ is selected from hydrogen (H),C₁-C₆ alkyl or C₁-C₆ alkenyl, wherein the alkyl or alkenyl moiety may besubstituted with one or more halogen atoms or (halo)alkoxy moieties,preferably R³ is H or C₁-C₆ alkyl, more preferably R³ is H or C₁-C₄alkyl, even more preferably R³ is H or C₁-C₂ alkyl, wherein the alkylmoiety may be substituted with one or more halogen atoms or (halo)alkoxymoieties. Halogen atoms include fluorine (F), chlorine (Cl), bromine(Br), iodine (I), and astatine (At), preferably the halogen atom isfluorine (F). Preferred alkoxy moieties include methoxy and ethoxy. Inhaloalkoxy moieties, at least one hydrogen atom of an alkoxy moiety isreplaced by a halogen atom, preferably by F. Suitable moieties for R³include, preferably are limited to, H, methyl (Me), trifluoromethyl(—CF₃), ethyl (Et), isopropyl (iPr), cyclopropyl (-cPr), methylenecyclopropyl (—CH₂cPr), n-propyl (n-Pr), 2,2,2-trifluoroethyl (—CH₂CF₃)and methoxymethyl (—CH₂OCH₃), more preferably R³ is H or methyl (Me),most preferably R³ is H. Alternatively, R³ is preferably C₁-C₄ alkyl,wherein the alkyl moiety may be substituted with one or more halogenatoms or (halo)alkoxy moieties, more preferably R³ is C₁-C₂ alkyl,wherein the alkyl moiety may be substituted with one or more halogenatoms or (halo)alkoxy moieties.

Distal nitrogen atom N* may be in neutral or in cationic form. In afirst preferred embodiment, the distal nitrogen atom is in neutral form,in which case both R⁴ and X are absent. In an alternative preferredembodiment, the distal nitrogen atom is in cationic form, in which caseboth R⁴ and X are present as defined herein.

In case the distal nitrogen atom is in cationic form, it formallyoriginates from protonation or alkylation, preferably protonation ormethylation of a trivalent nitrogen atom. The trivalent nitrogen atom ispreferably an amine moiety, either primary, secondary or tertiary, or animine moiety, either primary or secondary. The counter ion (X) of thecationic distal nitrogen atom is a negatively charged ion, preferably amonovalent negatively charged ion, more preferably an anion as indicatedherein below. The synthesis of the compounds of the invention does notneed to encompass the protonation or alkylation of an amine or iminenitrogen atom. The cationic distal nitrogen atom may also be formed viaa different route. As such, the cationic distal nitrogen atom only“formally” originates from the protonation or alkylation of an amine orimine nitrogen atom.

R⁴ is the substituent on the cationic distal nitrogen atom, whichoriginates from formal protonation or alkylation of the amine or iminemoiety. Thus, the compound according to this embodiment, in view of thepresence of the cationic nitrogen atom and X, is a salt, preferably apharmaceutically acceptable salt. Pharmaceutically acceptable salts arethose salts that are suitable to be administered as drugs orpharmaceuticals to humans and/or animals. The pharmaceuticallyacceptable salts of the amine or imine moiety of the compound accordingto the invention are known to those skilled in the art, and originatefrom formal treatment of the compound with an acid (protonation agent)or an alkylating agent. Suitable acids include organic acids orinorganic acids. Examples of inorganic acids include, but are notlimited to, hydrochloric acid (HCl), hydrobromic acid (HBr), hydroiodicacid (HI), sulphuric acid (H₂SO₄), nitric acid (HNO₃), trifluoroaceticacid (TFAH or CF₃CO₂H) and phosphoric acid (H₃PO₄). Examples of organicacids include, but are not limited to, formic acid, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, sulfonic acids and salicylicacid. When an acid as exemplified here is used to formally prepare thesalt, R⁴ is hydrogen, and the type of acid determines counter ion X.Alternatively, the salt can be formed by formal treatment with analkylating agent. Suitable alkylating agents include, but are notlimited to, C₁-C₆ alkyl halides (such as methyl iodide, ethyl iodide,propyl iodide, butyl chloride, butyl fluoride, butyl bromide), dimethylsulphate, dimethyl carbonate, methyl triflate, methyl fluorosulfonate,methyl chlorosulfonate, methyl methanesulfonate and methylbenzenesulfonate. The salt may be prepared by actual treatment of thenon-salt compound with an acid or alkylation agent, as indicated above,or via other means known in the art and/or exemplified further below.

R⁴ is selected from hydrogen (H) or C₁-C₆ alkyl, wherein the alkylmoiety may be substituted with one or more halogen atoms or (halo)alkoxymoieties, preferably R⁴ is H or C₁-C₄ alkyl, wherein the alkyl moietymay be substituted with one or more halogen atoms or (halo)alkoxymoieties, more preferably R⁴ is H or C₁-C₂ alkyl, wherein the alkylmoiety may be substituted with one or more halogen atoms or (halo)alkoxymoieties. Halogen atoms include fluorine (F), chlorine (Cl), bromine(Br), iodine (I), and astatine (At), preferably the halogen atom isfluorine (F). Preferred alkoxy moieties include methoxy and ethoxy. Inhaloalkoxy moieties, at least one hydrogen atom of an alkoxy moiety isreplaced by a halogen atom, preferably by F. Suitable moieties for R⁴include, preferably are limited to, H, methyl (Me), trifluoromethyl(—CF₃), ethyl (Et), isopropyl (iPr), cyclopropyl (-cPr), methylenecyclopropyl (—CH₂cPr), n-propyl (n-Pr), 2,2,2-trifluoroethyl (—CH₂CF₃),methoxymethyl (—CH₂OCH₃). Even more preferably R⁴ is H or methyl (Me),most preferably R⁴ is H.

X can be any anion, preferably a physiologically or pharmaceuticallyacceptable anion, more preferably a monovalent anion. X is preferablyselected from F, Cl, Br, I, HSO₄, NO₃, TFA (CF₃CO₂), formate, acetate,propionate, glycolate, pyruvate, oxalate, maleate, malonate, succinate,fumarate, tartarate, citrate, benzoate, cinnamate, mandelate, sulfonateand salicylate. Preferably, X is Cl, I, TFA or formate, more preferablyCl, I, TFA or formate, even more preferably X is Cl or formate, mostpreferably X is Cl. When the cationic nitrogen atom originates fromformal protonation, this protonation is preferably accomplished withhydrogen chloride (HCl), trifluoroacetic acid (TFAH or CF₃CO₂H) orformic acid (HCOOH), more preferably with HCl or formic acid. Formalmethylation is preferably accomplished with methyl iodide (MeI). Thus,in a preferred embodiment, R⁴=Me when X═I⁻, and R⁴═H when X═Cl⁻, TFA⁻ orformate.

In an especially preferred embodiment, the distal nitrogen atom isneutral and R² is joined with a backbone atom of the linker L in acyclic structure as further defined above, preferably a saturated cyclicstructure, most preferably a piperidine ring.

Appropriate linkers L to connect the amide nitrogen atom to the distalnitrogen atom are linkers preferably comprising 1 to 10 optionallysubstituted backbone atoms more preferably comprising 1 to 8 optionallysubstituted backbone atoms. L can thus comprise 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 optionally substituted backbone atoms. Herein, backbone atomsare those atoms that make up the shortest chain between the amidenitrogen atom and the distal nitrogen atom. The backbone may be a linearstructure, but may also be part of a cyclic structure. When the backboneis part a cyclic structure, the backbone is defined as the shortestchain between the amide nitrogen atom and the distal nitrogen atom. Insuch instances, one of the backbone atoms comprises a substituent R⁵,and one of the backbone atoms comprises a substituent R^(5′), preferablytwo different backbone atoms comprise the substituents R⁵ and R^(5′),wherein R⁵ and R^(5′) are joined to form a cyclic structure, preferablya 4-10-membered cyclic structure, more preferably a 5-8-membered cyclicstructure, most preferably a 6-membered cyclic structure. In thisembodiment, the amide nitrogen atom and the distal nitrogen atom are notincluded in the cyclic structure, but instead only part of the backboneof the linker is included. In a preferred embodiment, this connectionbetween the backbone atom(s) of the linker, bearing the R⁵ and R^(5′)substituents, is a —(CH₂)_(n)— bridge, wherein n=1-6, preferably a—CH₂—CH₂— or —CH₂—CH₂—CH₂— bridge, wherein one to six, preferably two orthree, carbon atoms are present between the substituted backbone atom(s)of the linker.

In a preferred embodiment, the backbone atoms are selected from carbon,nitrogen and oxygen, preferably from carbon and nitrogen. Such abackbone according to this preferred embodiment may be identified asC_(n-m)N_(m), wherein n designates the total number of atoms in thebackbone, and m the number of nitrogen atoms in the backbone. Each of nand m is a non-negative integer. Suitable linkers have n=1-10 and m=0-4,preferably n=2-7 and m=0-3, more preferably n=4-7 and m=0-2. Especiallypreferred linkers have a backbone identified as C_(n-m)N_(m), whereinn=2 and m=0 (C₂); n=5 and m=1 (C₄N); n=3 and m=0 (C₃); n=4 and m=1(C₃N); n=7 and m=2 (C₅N₂); n=4 and m=0 (C₄); n=6 and m=1 (C₅N); or n=5and m=0 (C₅). Most preferably, all backbone atoms are carbon atoms(m=0).

To fulfil their valence requirements, the carbon and nitrogen backboneatoms of the linker may bear hydrogen atoms, may be substituted, ordouble or triple bonds may be present between adjacent backbone atoms,as will be understood by the skilled person. In the context of theinvention, hydrogen is not regarded a substituent. Whenever an oxygenatom is present as backbone atom in the linker, the skilled person willunderstand that the oxygen backbone atom bears no hydrogen atoms,substituents or double or triple bonds. Triple bonds may be presentbetween two carbon atoms of the backbone. The backbone atoms, togetherwith the hydrogen atoms and/or the substituents, constitute the linker.In the context of the present invention, “optionally substituted” isused to indicate that an (backbone) atom may bear one or moresubstituents, or may bear no substituents and sufficient hydrogen may bepresent instead, to fulfil the valence requirements of said (backbone)atom.

Suitable substituents include but are not limited to halogen, NH₂, NHR⁶,N(R⁶)₂, NHNH₂, N₃, NHC(═O)R⁶, NHC(═O)NHR⁶, NHC(═O)NH₂, NHC(═O)OR⁶, OH,OR⁶, OC(═O)R⁶, R⁶ (e.g. alkyl, cycloalkyl), aralkyl, alkenyl, alkynyl,aryl, heteroaryl, OC(═O)OR⁶, OC(═O)NR⁶, O(SO₂)R⁶, O(SO₂)OH, O(PO₂)OH,SH, SR⁶, C(═O)R⁶, alkyl-NH₂, alkyl-OH, alkyl-SH, C(═O)CF₃, C(═O)OR⁶,C(═O)OH, C(═O)H, C(═O)OR⁶, C(═O)NH₂, C(═O)NMe₂, C(═O)N(R⁶)₂, C(═S)NH₂C(═S)SH, CN, NC, CNO, ONC, OCN, SCN, SNC, CNS, S(═O)R⁶, S(═O)₂R⁶,S(═O)₂(OH), P(═O)(OH)₂ or P(═O)(OH)(OR⁶). Atoms having two or moreremaining valencies, such as carbon backbone atoms, may bear a doublebonded substituent, such as oxo (═O), imino (═NH or ═NRW), thioxo (═S),alkylidene (═CH₂ or ═CHR⁶ or ═C(R⁶)₂). In addition, two substituents onthe same atom or on different atoms may be joined to form cyclicstructures. If two substituents on a single backbone atom are joined ina cyclic structure, this cyclic structure may be regarded as beingconnected via a spiro junction to the backbone. If two substituents ondifferent backbone atoms are joined in a cyclic structure, part of thiscyclic structure is (part of) the backbone, and the backbone isconsidered to be the shortest chain of atoms between the amide nitrogenatom and the distal nitrogen atom. As further indicated below, a cyclicstructure may also be formed by joining one substituent on a backboneatom with R¹ on the amide nitrogen atom or with R² on the distalnitrogen atom. The cyclic structures formed as such may be all-carbon ormay comprise 0-3 heteroatoms (e.g. N, O, S and/or P), and may comprise0-3 double bonds. All atoms in these cyclic structures may optionally besubstituted. Examples of suitable cyclic structures are optionallysubstituted cycloalkyl, optionally substituted cycloheteroalkyl,optionally substituted aryl or optionally substituted heteroaryl. Hereabove, each R⁶ is independently an alkyl moiety, preferably a C₁-C₆alkyl moiety, more preferably a C₁-C₂ alkyl moiety. Within R⁶, one ormore CH₂ moieties may each independently be replaced by one of O, S orNH, and/or one or more CH moieties may be replaced by N.

In the context of the present invention, the term “alkyl” refers tosaturated aliphatic groups including straight-chain, branched-chain,cyclic groups, and combinations thereof, having the number of carbonatoms specified, or if no number is specified, preferably having up to12 carbon atoms. “Straight-chain alkyl” or “linear alkyl” group refersto alkyl groups that are neither cyclic nor branched, commonlydesignated as “n-alkyl” groups. One subset of alkyl groups is C₁-C₆alkyl, which includes groups such as methyl, ethyl, n-propyl, isopropyl,butyl, n-butyl, isobutyl, sec-butyl, t-butyl, pentyl, n-pentyl, hexyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and any other alkylgroup containing between one and six carbon atoms, where the C₁-C₆ alkylgroups can be attached via any valence on the C₁-C₆ alkyl groups.

Preferred substituents of the backbone atoms are alkyl, such as methyl(Me or —CH₃), carboxy (—C(═O)OH), oxo (═O) and primary amino (—NH).

Preferred linkers are identified here below as L¹ to L²⁶:

L¹ =

—CH₂—CH₂— or —(CH₂)₂— L² =

—CH₂—CH₂—NH—C(O)—CH₂— or —(CH₂)₂NHC(O)CH₂— L³ =

—CH₂—CH₂—CH₂— or —(CH₂)₃— L⁴ =

—CH₂—CH₂—NH—C(NH₂)═ or —(CH₂)₂NHC(NH₂)═ L⁵ =

—CH₂—CH₂—NH—C(O)—CH₂—NH—C(NH₂)═ or —(CH₂)₂NHC(O)CH₂NHC(NH₂)═ L⁶ =

—CH₂—CH₂—CH₂—NH—C(NH₂)═ or —(CH₂)₃NHC(NH₂)═ L⁷ =

—CH₂—CH₂—NH—C(Me)═ or —(CH₂)₂NHC(Me)═ L⁸ =

—CH₂—CH₂—NH—C(O)—CH₂—NH—C(Me)═ or —(CH₂)₂NHC(O)CH₂NHC(Me)═ L⁹ =

—CH₂—CH₂—CH₂—NH—C(Me)═ or —(CH₂)₃NHC(Me)═ L¹⁰ =

—CH₂—CH₂—NR^(1′)—C(NH₂)═ or —(CH₂)₃NR^(1′)C(NH₂)═ L¹¹ =

—C(CO₂H)—CH₂—CH₂—CH₂— or —C(CO₂H)(CH₂)₃— L¹² =

—C(CO₂H)—CH₂—CH₂—CH₂—NH—C(NH₂)═ or —C(CO₂H)(CH₂)₃NHC(NH₂)═ L¹³ =

—C(CO₂H)—CH₂— or —C(CO₂H)CH₂— L¹⁴ =

—C(CO₂H)—CH₂—CH₂— or —C(CO₂H)(CH₂)₂— L¹⁵ =

—C(CO₂H)—CH₂—CH₂—CH₂—CH₂— or —C(CO₂H)(CH₂)₄— L¹⁶ =

—CH₂—CH₂—CH₂—CH₂— or —(CH₂)₄— L¹⁷ =

—CH₂—CH₂—CH₂—CH₂—CH₂— or —(CH₂)₅— L¹⁸ =

—CHR^(2′)—C(O)— or —CHR^(2′)C(O)  L¹⁹ =

—CHR^(2′)—CH₂— or —CHR^(2′)CH₂— L²⁰ =

—CHR⁵—CH₂—NR^(5′)—C(Me)═ or —CHR⁵CH₂NR^(5′)C(Me)═ L²¹ =

—CHR^(2′)—CH₂—CH₂— or —CHR^(2′)(CH₂)₂— L²² =

—CH₂—CH₂—CHR^(1′)— or —(CH₂)₂CHR^(1′)— L²³ =

—CH₂—CH₂—CHR^(1′)—NH—C(O)—C(Me)— or —(CH₂)₂CHR^(1′)NHC(O)C(Me)— L²⁴ =

—CH₂—CHR^(1′)— or —CH₂CHR^(1′)— L²⁵ =

—CH₂—CHR^(1′)—NH—C(Me)═ or —CH₂CHR^(1′)NHC(Me)═ L²⁶ =

—CHR⁵—CH₂—CH₂—CHR^(5′)— or —CHR⁵(CH₂)₂CHR^(5′)—

Herein, the dashed bond at the left side of each of the structures forL¹ to L²⁶ indicates the bond between the linker and the amide nitrogenatom, and the dashed bond at the right side of each of the structuresfor L¹ to L²⁶ indicates the bond between the linker and the distalnitrogen atom. The linkers depicted as chemical formulas are oriented inthe same direction, i.e. the pendant bond at the left side of each ofthe chemical formulas for L¹ to L²⁶ indicates the bond between thelinker and the amide nitrogen atom, and the dashed bond at the rightside of each of the chemical formulas for L¹ to L²⁶ indicates the bondbetween the linker and the cationic nitrogen atom.

Each occurrence of R^(1′) represents the connection of a second linkerbetween the linker and the amide nitrogen atom, wherein R^(1′) is joinedwith R¹ via said bridge, thus forming a 4-10-membered cyclic structure,preferably a 5-8-membered cyclic structure, most preferably a 6-memberedcyclic structure, which is built up from the amide nitrogen atom, 1-4atoms of the backbone of the linker, and 1-4 atoms which make up thebridge joining R¹ and R^(1′). Likewise, each occurrence of R^(2′)represents the connection of a second linker between the linker and thecationic nitrogen atom, wherein R^(2′) is joined with R² via saidbridge, thus forming a 4-10-membered cyclic structure, preferably a5-8-membered cyclic structure, most preferably a 6-membered cyclicstructure, which is built up from the cationic nitrogen atom, 1-4 atomsof the backbone of the linker, and 1-4 atoms which make up the bridgejoining R² and R^(2′). Likewise, each occurrence of R⁵ and R^(5′)represent the connection of a second linker between one backbone atom ofthe linker, bearing R⁵, and another backbone atom of the linker, bearingR^(5′), wherein R^(5′) is joined with R⁵ via said bridge, thus forming a4-10-membered cyclic structure, preferably a 5-8-membered cyclicstructure, most preferably a 6-membered cyclic structure, which is builtup from 2-5 atoms of the backbone of the linker, and 1-5 atoms whichmake up the bridge joining R⁵ and R^(5′). Thus, in linkers L¹⁰, L²²,L²³, L²⁴ and L²⁵, R^(1′) is joined to R¹ via a second linker, preferablya —CH₂—CH₂— or —CH₂—CH₂—CH₂— bridge, more preferably a —CH₂—CH₂-bridge.Thus, in a compound comprising linker L¹⁰, wherein R^(1′) and R¹ arejoined via a —CH₂—CH₂— bridge, the amide nitrogen atom is embedded in asix-membered cyclic structure, which is built up from the amide nitrogenatom, two carbon atoms and one nitrogen atom of the backbone of thelinker, and two more carbon atoms which make up the bridge of R¹ andR^(1′). This —CH₂—CH₂— bridge between the amide nitrogen atom and thecentral nitrogen atom in the backbone of linker L¹⁰ may be representedas L. Likewise, in linkers L¹⁸, L¹⁹ and L²¹, R² is joined to R² via asecond linker, preferably a —CH₂—CH₂— or —CH₂—CH₂—CH₂— bridge, morepreferably a —CH₂—CH₂—CH₂— bridge. Likewise, in linker L²⁰ and L²⁶,R^(5′) is joined to R⁵ via a second linker, preferably a —CH₂—CH₂— or—CH₂—CH₂—CH₂— bridge, more preferably a —CH₂—CH₂— bridge.

Linkers L¹¹, L¹², L¹³, L¹⁴, L¹⁵, L¹⁶ (as long as R²-R^(2′) is not—C(O)—), L¹⁹ (as long as R²-R^(2′) is not —CH₂—), L²⁰ (as long asR⁵-R^(5′) is not —CH₂—), L^(2′) (as long as R²-R^(2′) is not —CH₂—CH₂—),L²² (as long as R¹-R^(1′) is not —CH₂—CH₂—), L²³ (as long as R¹-R^(1′)is not —CH₂—CH₂—), L²⁴ (as long as R¹-R^(1′) is not —CH₂—) and L²⁵ (aslong as R¹-R^(1′) is not —CH₂—) comprise an additional stereocenter. Thestereoisomer, when indicated in the structures of those linkers, aboveis meant as illustrative, not as limiting. As indicated further above,each stereocenter present in the compounds according to the inventionmay individually be present in each of its stereoisomeric forms, eitherS or R, or as a mixture of both isomers in any ratio. Linker L²⁶comprises a disubstituted cycloalkyl moiety, preferably a disubstitutedcyclohexyl moiety, and may thus occur in either the cis-form or thetrans-form, preferably in the trans-form.

Especially preferred linkers are L⁵, L⁸, L¹¹, L¹², L¹⁶, L¹⁷, L¹⁹, L²¹and L²⁶. Even more preferred linkers are L, L¹⁶, L¹⁹ and L²⁶, and mostpreferably the linker is L¹⁹. Preferably, L¹⁹ is combined withR²-R^(2′)=L¹ or L³, most preferably with R²-R^(2′)=L³. Preferably, L²¹is combined with R²-R^(2′)=L¹ or L³, most preferably with R²-R^(2′)=L¹.Preferably, L²⁶ is combined with R⁵-R^(5′)=L¹ or L³, more preferablywith R⁵-R^(5′)=L¹, most preferably wherein the cyclohexyl istrans-1,4-disubstituted. Especially preferred is the combination oflinker L¹⁹ with R²-R^(2′)=L³ and R³═H, Me, Et, iPr, CH₂OCH₃ or CH₂CF₃,more preferably R³=Me, Et, iPr or CH₂CF₃, most preferably R³═H

In case R⁴ and X are absent, it is preferred that linker L contains 1-5optionally substituted backbone atoms and/or linker L contains at leastone backbone atom other than carbon. In case R⁴ and X are absent, it isespecially preferred that the distal nitrogen atom is connected to abackbone atom of the linker via a second linker wherein R² is joinedwith R^(2′), more preferably wherein the cyclic structure thus formed isa piperidine ring, a pyrrolidine ring, an imidazolidine ring, apyrazolidine ring or an azepane ring, most preferably a piperidine ring,and/or at least one of the backbone atoms is substituted with acarboxylic acid moiety. In case R⁴ and X are absent, it is especiallypreferred that L is any one of L², L⁴-L²¹, L²³, L²⁵ and L²⁶, morepreferably one of L⁵, L⁸, L¹¹, L¹², L¹⁶, L¹⁷, L¹⁹, L²¹ and L²⁶.

In case R⁴ and X are present, it is preferred that R⁴ is H or Me, morepreferably R⁴ is H, and X is Cl, I, TFA or formate, even more preferablyX is Cl or formate, most preferably X is Cl. In case R⁴ and X arepresent, it is preferred that linker L contains 3-10 backbone atoms, or2 backbone atoms of which one is connected to the distal nitrogen atomvia a second linker. In case R⁴ and X are present, it is especiallypreferred that L is any one of L²-L²⁶, more preferably one of L⁵, L⁸,L¹¹, L¹², L¹⁶, L¹⁷, L¹⁹, L²¹ and L²⁶.

In one preferred embodiment, the compound according to the invention isrepresented by general structure (I), wherein

-   -   L is a linker between the amide nitrogen atom and the distal        nitrogen atom;    -   R¹ is selected from hydrogen (H), C₁-C₆ alkyl or C₁-C₆ alkenyl,        or R¹ is joined with a backbone atom of the linker L in a cyclic        structure;    -   R² is joined with a backbone atom of the linker L to form a        cyclic structure selected from a piperidine ring, a pyrrolidine        ring, an imidazolidine ring, a pyrazolidine ring or an azepane        ring;    -   R³ is selected from hydrogen (H), C₁-C₆ alkyl or C₁-C₆ alkenyl,        wherein the alkyl or alkenyl moiety may be substituted with one        or more halogen atoms or (halo)alkoxy moieties, or R³ is absent        when the distal nitrogen atom is part of an imine moiety; and    -   R⁴ and X are absent.

In an alternative preferred embodiment, the compound according to theinvention is represented by general structure (I), wherein

-   -   L is a linker between the amide nitrogen atom and the distal        nitrogen atom comprising 3-10 backbone atoms, or 2 backbone        atoms of which one is connected to the distal nitrogen atom via        a second linker;    -   R¹ and R² are each independently selected from hydrogen (H),        C₁-C₆ alkyl or C₁-C₆ alkenyl, or R¹ and R² are joined together        and thus form a second linker between the amide nitrogen atom        and the distal nitrogen atom, or R¹ is joined with a backbone        atom of the linker L in a cyclic structure and/or R² is joined        with a backbone atom of the linker L in a cyclic structure;    -   R³ is selected from hydrogen (H), C₁-C₆ alkyl or C₁-C₆ alkenyl,        wherein the alkyl or alkenyl moiety may be substituted with one        or more halogen atoms or (halo)alkoxy moieties, or R³ is absent        when the distal nitrogen atom is part of an imine moiety;    -   R⁴ is selected from hydrogen (H) or C₁-C₆ alkyl, wherein the        alkyl moiety may be substituted with one or more halogen atoms        or (halo)alkoxy moieties; and    -   X is an anion, preferably a pharmaceutically acceptable anion.

Preferred compounds according to the invention are identified here belowas compounds A to AH, which are defined by general structure (I),wherein:

-   -   for compound A: L=L¹, R¹-R²=L¹, R³═H;    -   for compound B: L=L¹, R¹═H, R²═H, R³═H;    -   for compound C: L=L², R¹═H, R²═H, R³═H;    -   for compound D: L=L³, R¹═H, R²═H, R³═H;    -   for compound E: L=L⁴, R¹═H, R²═H, R³═absent;    -   for compound F: L=L⁵, R¹═H, R²═H, R³═absent;    -   for compound G: L=L⁶, R¹═H, R²═H, R³═absent;    -   for compound H: L=L³, R¹═H, R²=Me, R³=Me;    -   for compound I: L=L¹, R¹═H, R²=Me, R³=Me;    -   for compound J: L=L⁷, R¹═H, R²═H, R³═absent;    -   for compound K: L=L⁸, R¹═H, R²═H, R³═absent;    -   for compound L: L=L⁹, R¹═H, R²═H, R³═absent;    -   for compound M: L=L¹⁰, R¹-R^(1′)=L¹, R²═H, R³═absent;    -   for compound N: L=L¹¹, R¹═H, R²═H, R³═H;    -   for compound O: L=L¹², R¹═H, R²═H, R³═absent;    -   for compound P: L=L¹³, R¹═H, R²═H, R³═H;    -   for compound Q: L=L¹⁴, R¹═H, R²═H, R³═H;    -   for compound R: L=L¹⁵, R¹═H, R²═H, R³═H;    -   for compound S: L=L¹¹, R═H, R²=Me, R³=Me    -   for compound T: L=L¹⁶, R═H, R²═H, R³═H;    -   for compound U: L=L¹⁷, R═H, R²═H, R³═H;    -   for compound V: L=L¹⁶, R═H, R²=Me, R³=Me;    -   for compound W: L=L¹⁸, R¹═H, R²-R^(2′)=L³, R³═H;    -   for compound X: L=L¹⁹, R¹═H, R²-R^(2′)=L³, R³═H;    -   for compound Y: L=L²⁰, R═H, R²═H, R⁵-R^(5′)=L³, R³═absent;    -   for compound Z: L=L²¹, R¹═H, R²-R^(2′)=L¹, R³═H;    -   for compound AA: L=L²², R¹-R^(1′)=L, R²═H, R³═H;    -   for compound AB: L=L²³, R¹-R^(1′)=L¹, R²═H, R³═H;    -   for compound AC: L=L²⁴, R¹-R^(1′)=L³, R²═H, R³═H;    -   for compound AD: L=L²⁵, R¹-R^(1′)=L³, R²═H, R³═absent;    -   for compound AE: L=L²⁶, R¹═H, R²═H, R⁵-R^(5′)=L¹, R³═H.    -   for compound AF: L=L¹⁹, R¹═H, R²-R^(2′)=L³, R³=Me;    -   for compound AG: L=L¹⁹, R¹═H, R²-R^(2′)=L¹, R³═H;    -   for compound AH: L=L²¹, R¹═H, R²-R^(2′)=L¹, R³=Me.

Herein, R⁴ and X may either be present or absent. In case R⁴ and X areabsent, it is especially preferred that the compound according to theinvention is selected from compounds A-F and J-AH. In case R⁴ and X arepresent, it is especially preferred that the compound according to theinvention is selected from compounds A-H and J-AH, more preferablywherein R⁴═H and X═Cl or formate, most preferably wherein R⁴═H and X═Cl.

Especially preferred compounds according to the invention are compoundsF, K, N, O, U, V, T, X, Z, AE, AF, AG and AH, more preferably N, T, Xand AE, most preferably X.

Compound F may have the R-configuration, the S-configuration or amixture thereof, preferably compound F is a mixture of the R- andS-enantiomers, more preferably a racemic mixture. Compound K may havethe R-configuration, the S-configuration or a mixture thereof,preferably compound K is a mixture of the R- and S-enantiomers, morepreferably a racemic mixture. Compound N may have the R,R-configuration,R,S-configuration, S,R-configuration, the S,S-configuration or anymixture thereof, preferably compound N has the R,R-configuration or theS,R-configuration, most preferably the R,R-configuration. Compound O mayhave the R,R-configuration, R,S-configuration, S,R-configuration, theS,S-configuration or any mixture thereof, preferably compound O is amixture of the R,S- and S,S-diastereomers more preferably about 1/1(mol/mol) mixture. Compound U may have the R-configuration, theS-configuration or a mixture thereof, preferably compound U has theR-configuration or the S-configuration. Compound V may have theR-configuration, the S-configuration or a mixture thereof, preferablycompound V has the R-configuration. Compound T may have theR-configuration, the S-configuration or a mixture thereof, preferablycompound T has the R-configuration or the S-configuration, mostpreferably the R-configuration. Compound X may have theR,R-configuration, R,S-configuration, S,R-configuration, theS,S-configuration or any mixture thereof, preferably compound X has theR,S-configuration or the S,R-configuration, most preferably theS,R-configuration. Compound Z may have the R-configuration, theS-configuration or a mixture thereof, preferably compound Z is a mixtureof the R- and S-enantiomers, more preferably a racemic mixture. CompoundAE may have the R,trans-configuration, R,cis-configuration,S,trans-configuration, the S,cis-configuration or any mixture thereof,preferably compound AE has the R,trans-configuration or theS,trans-configuration, most preferably the R,trans-configuration.Compound AF may have the R,R-configuration, R,S-configuration,S,R-configuration, the S,S-configuration or any mixture thereof,preferably compound AF has the S,R-configuration. Compound AG may havethe R,R-configuration, R,S-configuration, S,R-configuration, theS,S-configuration or any mixture thereof, preferably compound AG has theS,S-configuration or the S,R-configuration. Compound AH may have theR-configuration, the S-configuration or a mixture thereof, preferablycompound AH has the S-configuration. Herein, the first designator (R orS) of the configuration is for the 2-position of the open-Trolox moiety,and in case an additional stereocenter is present in the compoundaccording to the invention, the second designator thereof defines theconfiguration.

The most preferred compounds according to the invention are compound Nin the R,R-configuration (R,R—N), compound T in the R-configuration(R-T), compound AE in the R,trans-configuration (R,trans-AE) andcompound X in any configuration, most preferably the compound accordingto the invention is compound X in the S,R-configuration (S,R—X). In oneembodiment, these most preferred compounds according to the inventionare compound N in the R,R-configuration (R,R—N), compound T in theR-configuration (R-T), compound AE in the R,trans-configuration(R,trans-AE) and compound X in any configuration, wherein R⁴═H and X═Cl,more preferably the compound according to the invention is compound X inthe S,R-configuration (S,R—X), wherein R⁴═H and X═Cl. In one embodiment,these most preferred compounds according to the invention are compound Nin the R,R-configuration (R,R—N), compound T in the R-configuration(R-T), compound AE in the R,trans-configuration (R,trans-AE) andcompound X in any configuration, wherein R⁴ and X are absent, mostpreferably the compound according to the invention is compound X in theS,R-configuration (S,R—X), wherein R⁴ and X are absent.

The invention also includes all stereoisomers and geometric isomers ofthe compounds, including diastereomers, enantiomers, and cis/trans (E/Z)isomers. The invention also includes mixtures of stereoisomers and/orgeometric isomers in any ratio, including, but not limited to, racemicmixtures.

In one embodiment, the compound according to the invention is not thecompound represented by structure (I), wherein:

-   -   L=—C(iPr)-C(O)—NH—C(4-chlorobenzyl)-C(O)—C(O)—, R¹═H,        R²═—CH₂—CH₃ (Et), R³═H, R⁴═X=absent; and/or    -   L=—(CH₂)₂—NR¹-pPh-N═C(thiophen-2-yl)-, R¹-R^(1′)═—(CH₂)₂-(L¹),        R²═H, R³═H; R⁴═X=absent, wherein pPh represents a        para-substituted phenylene ring; and/or    -   L=—(CH₂)₂-(L¹), R¹═H, R²=Me, R³=Me, R⁴═X=absent; and/or    -   L=—CH₂—C_(p)(R²)—(C_(p)H)₂—, R¹═H, R²-R^(2′)═—(C_(p)H)₂—,        R³═R⁴═X=absent, wherein all instances of C_(p) together with the        distal nitrogen atom make up a pyridine ring; and/or    -   L=—CH₂—C_(p)(R²)—C_(p)H—, R¹═H, R²-R^(2′)═—(C_(p)H)₃—,        R³═R⁴═X=absent, wherein all instances of C_(p) together with the        distal nitrogen atom make up a pyridine ring; and/or    -   L=—CH₂—C_(p)(R²)—, R¹═H, R²-R^(2′)═—(C_(p)H)₄—, R³═R⁴═X=absent,        wherein all instances of C_(p) together with the distal nitrogen        atom make up a pyridine ring; and/or    -   L=—(CH₂)₃-(L³), R¹═H, R²=Me, R³=Me, R⁴═X=absent; and/or    -   L=—(CH₂)₂-(L¹), R¹-R²═—(CH₂)₂-(L¹), R³=Me, R⁴═X=absent; and/or    -   L=—(CH₂)₂-(L¹), R¹-R²═—(CH₂)₂-(L¹), R³═H, R⁴═X=absent; and/or    -   L=—(CH₂)₂—C_(p)(R²)—, R¹═H, R²-R^(2′)═—(C_(p)H)₄—,        R³═R⁴═X=absent, wherein all instances of C_(p) together with the        distal nitrogen atom make up a pyridine ring; and/or    -   L=—(CH₂)₃—N_(i)(R²)—C_(i)H—, R¹═H, R²-R^(2′)═—(C_(i)H)₂—,        R³═R⁴═X=absent, wherein all instances of C_(i) together with        N_(i) and the distal nitrogen atom make up an imidazole ring;        and/or    -   L=—(CH₂)₂-(L¹), R¹-R²═—(CH₂)₂-(L¹), R³═iPr, R⁴═X=absent; and/or    -   L=—C(R⁵)═C(R⁵)—, R¹═H, R²═H, R³═H, R⁴═X=absent,        R⁵-R⁵═—C(O)—N(Me)-C(O)—N(Ph)-, wherein linker R⁵-R⁵ is bound via        the C(O) moiety to the carbon atom adjacent the amide nitrogen        atom and via the N(Ph) moiety to the carbon atom adjacent the        distal nitrogen atom; and/or    -   L=—(CH₂)₂-(L¹), R¹═H, R²=Me, R³=Me, R⁴═H; X═Cl; and/or    -   L=—(CH₂)₂-(L¹), R¹═H, R²=Me, R³=Me, R⁴═H; X=MeSO₃.

The compounds can be administered in prodrug form. Prodrugs arederivatives of the compounds which are themselves relatively inactive,but which convert into the active compound when introduced into thesubject in which they are used, by a chemical or biological process invivo, such as an enzymatic conversion. Suitable prodrug formulationsinclude, but are not limited to peptide conjugates of the compounds ofthe invention and esters of compounds of the inventions. Furtherdiscussion of suitable prodrugs is provided in H. Bundgaard, Design ofProdrugs (New York: Elsevier, 1985); in R. Silverman, The OrganicChemistry of Drug Design and Drug Action (Boston: Elsevier, 2004); in R.L. Juliano, Biological Approaches to the Controlled Delivery of Drugs(Annals of the New York Academy of Sciences, volume 507, New York: N. Y.Academy of Sciences, 1987); and in E. B. Roche, Design ofBiopharmaceutical Properties Through Prodrugs and Analogs (Symposiumsponsored by Medicinal Chemistry Section, APhA Academy of PharmaceuticalSciences, November 1976 national meeting, Orlando, Fla.), published byThe Academy in Washington, 1977.

The various compounds of the invention can be administered either astherapeutic or cosmetic agents in and of themselves, or as prodrugswhich will convert to other effective substances in the body.Preferably, the compounds according to the invention are administered assuch, i.e. not as prodrug.

The compounds of the invention are useful for modulating ROS levels, aswell as for modulating mitochondrial morphology, i.e. eithermitochondrial fragmentation or mitochondrial filamentation, and/or formodulating the expression (i.e. steady-state levels) of OXPHOS enzymes,such as complex I and complex II. Thus, in one aspect, the inventionrelates to the use of the compounds of the invention in therapeuticand/or cosmetic methods for modulating at least one of mitochondrialmorphology, expression of OXPHOS enzymes and ROS levels, preferably formodulating ROS levels.

In one embodiment, the effect of the compounds of the invention includesone or more of induction of mitochondrial filamentation, prevention orreduction of mitochondrial fragmentation, and increased expression ofOXPHOS enzymes. Preferred compounds for this embodiment are compoundswherein the linkers are L⁵, L⁸, L¹¹, L¹², L¹⁶, L¹⁷, L¹⁹, L²¹ and L²⁶,preferably in these compounds the linkers are L, L¹⁶, L¹⁹ and L²⁶, andmost preferred the linkers according to this embodiment of the inventionis L¹⁹. More specifically, preferred compounds of the invention havingone or more of these effects are compounds F, K, N, O, U, V, T, X, Z,AE, AF, AG and AH. More preferred compounds according to the inventionhaving one or more of these effects are compounds N T, X and AE. Themost preferred compound according to this embodiment of the invention iscompound X.

Compounds according to the invention can for example be prepared fromcompounds of general structure (II) by means known in the art.

Herein, L, R¹, R², R³, R⁴ and X are as defined for the compounds ofgeneral structure (I). Compounds of general structure (II), and thesynthesis thereof, are known from WO 2014/011047. The skilled personfinds further guidance for preparing the compounds according to theinvention in Example 1.

In another aspect, the invention relates to a compound of the inventionas herein defined above for use as a medicament. The medicament can beused for both medical (human) as well as veterinary (animal)applications.

In a further aspect, the invention pertains to method of treating,preventing, or suppressing symptoms associated with a pathology,condition or disorder associated with an increased or an decreased ROSlevel, the method comprising administering to a subject an effectiveamount of one or more compounds of the invention as defined hereinabove. Alternatively, the invention relates to a compound as definedherein for use in treating, preventing or suppressing symptomsassociated with a pathology, condition or disorder associated with anincreased or an decreased ROS level. Preferably, the compound as definedherein can be used in treating, preventing or suppressing symptomsassociated with a pathology, condition or disorder associated with anincreased ROS level. The methods of the invention preferably compriseadministering to a subject an effective amount of one or more compoundsof the invention as herein defined above, and an acceptable carrier,excipient or vehicle, preferably a pharmaceutically or physiologicallyacceptable carrier, excipient or vehicle.

An increase in ROS levels causes oxidative stress, and the terms“increased ROS levels” and “oxidative stress” can be usedinterchangeable herein. An increased ROS level/oxidative stress isherein further defined as the imbalance between the systemicmanifestation of reactive oxygen species and the system's ability todetoxify the intermediates, or to repair the resulting damage. ROS(reactive oxygen species) are chemically reactive molecules containingoxygen, such as e.g. hydroxyl radical (OH), hydrogen peroxide (H₂O₂),superoxide radical (.O₂ ⁻). They are produced intracellularly throughmultiple mechanisms depending on the cell and tissue type.Alternatively, ROS can be generated by exogenous sources such asionizing radiation. The major source of intracellular ROS comes fromNAD(P)H oxidase complexes that are present in cell membranes,mitochondria, peroxisomes, and endoplasmic reticulum. For example, ROSare produced in the mitochondria as byproducts of normal cellrespiration (Kirkinezos IG and Moraes C T, Semin Cell Dev Biol.;12(6):449-457). Other sources of ROS include mitochondrial electrontransport enzymes, xanthine oxidase, cyclooxygenase, lipoxygenase, anduncoupled nitric oxide synthase.

ROS play an important role in may physiological processes such as hostdefence, cell signalling, hormone biosynthesis, fertilization andhomeostasis. However when intracellular ROS levels become too high,significant damage to cell structures may occur. The most common harmfuleffects of reactive oxygen species include DNA damage, oxidation ofpolyunsaturated fatty acids in lipids (lipid peroxidation), oxidation ofamino acids in proteins and oxidatively deactivate specific enzymes byoxidation of co-factors.

Consequently, increased ROS levels have been linked to either theprimary or secondary pathophysiologic mechanisms of acute and chronichuman diseases, such as Sickle cell disease, Alzheimer disease, Spinalcord injury, Systemic lupus erythematosus, Asthma, Systemic sclerosis(scleroderma), Unstable angina, Cutaneous leishmaniasis, Zellwegersyndrome, Preeclampsia, ARDS, Alcoholic liver disease, Atherosclerosis,Asbestosis, Ataxia telangiectasia, ALS, Cancer, Mild cognitiveimpairment, Diabetes mellitus (both types), HIV, Idiopathic pulmonaryfibrosis, Acute and chronic alcoholic liver disease, Ischemic brain,Parkinson disease, Acute chest syndrome of sickle cell disease,Respiratory distress syndrome, Retinopathy of prematurity, Wernersyndrome, Cardiopulmonary bypass, Cataract genesis, Chronic kidneydisease, COPD, Friedreich ataxia, HIV infections, Creutzfeldt-Jakobdisease, Hyperlipidemia, Renal cell carcinoma, Spherocytosis, Uremiaassociated with hemodialysis or peritoneal dialysis, Down syndrome,Heart failure, Hepatic cirrhosis, Huntington disease,Hypercholesterolemia, Hyperhomocysteinemia, Ischemia/Reperfusion injury,Interstitial lung disease, Myocardial infarction, Obesity, Crohndisease, Osteoporosis, Pancreatitis, Primary biliary cirrhosis,Psoriatic arthritis, Pulmonary hypertension, Lung injury, Reactivearthritis, Multiple sclerosis, Myocardial inflammation, Osteoarthritis,Rheumatoid arthritis, Severe bronchopulmonary dysplasia in neonates,Synucleinopathies, Tauopathies, Cardiovascular disease, Coronary arterydisease, End-stage renal disease, Aceruloplasminemia, Acute autoimmunemyocarditis, Acute pancreatitis, Bronchopulmonary dysplasia,Cataractogenesis, Chronic fatigue syndrome, Chronic hepatitis C, Chronicrenal failure, Cystic fibrosis, Diabetes (types 1 and 2), Helicobacterpylori infection and inflammation, Juvenile chronic arthritis, Lungcancer, Meningitis, Progeria, Psoriasis, Sarcoidosis, Sepsis, Systemicamyloidosis, Uremia (Dalle-Donne et al, Clinical Chem. (2006), 52(4):601-623).

In an embodiment of the invention, the compound as described herein canbe used for the treatment, prevention or suppression of symptoms of anyone of the diseases/conditions/pathologies listed herein above.

In a further preferred embodiment, the invention relates to a compoundfor use in treating, preventing, or suppressing symptoms associated withParkinson Disease, Alzheimer Disease, Epilepsy, Dementia, Asthma,Amyotrophic Lateral Sclerosis, Inborn metabolism errors, Systemicsclerosis, Atherosclerosis, Osteoarthritis, Rheumatoid arthritis,Xenobiotics toxicity, Post-ischemic Reperfusion injury, Hypertension,Pulmonary hypertension, Pulmonary edema, Bowel disease, Endometriosis,Diabetes, Diabetes-induced cardiac dysfunction, Diabetic nephropathy,Cancer, Embryonal rhabdomyosarcoma, congenital muscular dystrophies,dystrophinopathies Adrenoleukodystrophy, a mitochondrial disorder and acondition associated with mitochondrial dysfunction.

In a further preferred embodiment, the inborn metabolism error is alysosomal storage disorder, such as Gaucher's disease or Batten disease,and/or the inborn metabolism error is a disorder of peroxisomalfunction, such as Zellweger syndrome.

Reactive oxygen species is the general name for a number of reactivemolecules and free radicals derived from oxygen. In particular, reactiveoxygen species include superoxide, hydrogen peroxide, hydroxyl radical,hydroxyl ion and nitric oxide. Superoxide is the primary reactive oxygenspecies from which the others originate during detoxification bysuperoxide dismutase (SOD). The compounds of the invention may beparticularly useful for decreasing the level of intracellularsuperoxide. The superoxide level may be decreased towards homeostaticlevels and/or may be decreased below homeostatic levels. In particular,the superoxide levels in cell may be decreased even when there are noaberrant superoxide levels, e.g. the superoxide levels are not abovehomeostatic levels. Such decrease of super oxide may be particularlyuseful if a reactive oxygen species derived from super oxidedetoxification is increased above homeostatic levels.

In a particular embodiment, the invention relates to a compound asdefined herein for use in treating, preventing or suppressing symptomsassociated with of a pathology, condition or disorder associated with anincreased superoxide levels. Increased superoxide levels may originatefrom defects in enzymes that produce superoxide (e.g. NADPH oxidases)and/or enzymes involved in the detoxification of superoxides (e.g. SODs,catalases and/or peroxidases). A pathology, condition or disorder linkedto defects in enzymes that produce superoxide include, but is notlimited to, mitochondrial diseases (e.g. mitochondrial Complex I or IIIdeficiency), hypertension, diabetes, atherosclerosis, cardiovasculardisease and neurodegeneration (Paravicini™ et al, Diabetes Care 2008:S170-80). A pathology, condition or disorder linked to a dysfunction ofenzymes responsible for the detoxification of superoxide includes, butis not limited to amyotrophic lateral sclerosis (ALS) (Muyderman H et alBr. J Pharmacol 2014; 171(8):2191-205).

In a particularly preferred embodiment, the invention relates to acompound as defined herein for use in treating, preventing orsuppressing symptoms associated with an pathology, condition or disorderassociated with an increased ROS level, wherein the disorder is amitochondrial disorder and/or wherein the condition is a conditionassociated with mitochondrial dysfunction. In a further preferredembodiment, the mitochondrial disorder is a disorder selected from thegroup consisting of Myoclonic epilepsy; Myoclonic Epilepsy with RaggedRed Fibers (MERRF); Leber's Hereditary Optic Neuropathy (LHON);neuropathy ataxia and retinitis pigmentosa (NARP); MitochondrialMyopathy, Encephalopathy, Lactacidosis, Stroke (MELAS); Leigh syndrome;Leigh-like syndrome; Dominant Optic atrophy (DOA); Kearns-Sayre Syndrome(KSS); Maternally Inherited Diabetes and Deafness (MIDD);Alpers-Huttenlocher syndrome; Ataxia Neuropathy spectrum; ChronicProgressive External Ophthalmoplegia (CPEO); Pearson syndrome;Mitochondrial Neuro-Gastro-Intestinal Encephalopathy (MNGIE); Sengerssyndrome; 3-methylglutaconic aciduria, sensorineural deafness,encephalopathy and neuro-radiological findings of Leigh-like syndrome(MEGDEL); myopathy; mitochondrial myopathy; cardiomyopathy;encephalomyopathy, SURF1 (COX deficient Leigh syndrome due to complex IVsurfeit protein deficiency) and isolated or combined OXPHOS deficiencieswith so far unsolved genetic defect including disturbed pyruvateoxidation and ATP plus PCr production rates. In another preferredembodiment, the condition associated with mitochondrial dysfunctionpreferably is a condition selected from the group consisting ofFriedreich's Ataxia (FRDA); renal tubular acidosis; Parkinson's disease;Alzheimer's disease; amyotrophic lateral sclerosis (ALS); Huntington'sdisease; developmental pervasive disorders; hearing loss; deafness;congenital muscular dystrophies, dystrophinopathies, diabetes; ageing;and adverse drug effects hampering mitochondrial function.

In a further aspect, the invention relates to a method of treating,preventing, or suppressing symptoms associated with a mitochondrialdisorder or with a condition associated with mitochondrial dysfunction,the method comprising administering to a subject an effective amount ofone or more compounds of the invention as herein defined above.Alternatively, the invention relates to a compound of the invention asherein defined above, for use in a method of treating, preventing, orsuppressing symptoms associated with a mitochondrial disorder or with acondition associated with mitochondrial dysfunction. The methods of theinvention preferably comprise administering to a subject an effectiveamount of one or more compounds of the invention as herein definedabove, and an acceptable carrier, excipient or vehicle, preferably apharmaceutically or physiologically acceptable carrier, excipient orvehicle.

Preferred compounds of the invention for treating a mitochondrialdisorder and/or a condition associated with mitochondrial dysfunctionare compounds of which the effect includes one or more of induction ofmitochondrial filamentation, prevention or reduction of mitochondrialfragmentation, increased expression of OXPHOS enzymes, and decreased ROSlevels, preferably decreased ROS levels, as defined herein above.

In the methods of the invention, the mitochondrial disorder and/or thecondition associated with mitochondrial dysfunction preferably is acondition characterised by oxidative stress and/or a conditioncharacterized by an OXPHOS deficiency. Every cell needs energy. Shortageof energy therefore affects the activity of every cell. Thus inprinciple every cell is affected by a sub-optimal amount of one or moreof the OXPHOS complexes. However, the actual amount that is sub-optimalvaries from cell to cell. Cells that have a relatively high energyconsumption such as brain and muscle cells typically require a higheramount of OXPHOS system complexes than cells that have a low energyconsumption, such as resting T-cells. Thus, the cells that are affectedby said deficiency associated with an oxidative phosphorylationdeficiency are typically, but not necessarily muscle cells or braincells. Mitochondrial disorders are pleiotropic in their clinicalmanifestation. Various tissues can be affected like for instancepancreas, heart, liver, eye, inner ear, blood, colon and kidney. Inaddition, also cells from non-clinically affected tissues likefibroblasts often show a mitochondrial defect. Cells affected by anOXPHOS deficiency can be treated and provided with a higher amount ofOXPHOS complex by providing the cell with a compound of the invention. Acell is affected by an OXPHOS deficiency when the OXPHOS capacity islower than normal (i.e. a comparable cell of the same species from ahealthy individual). The capacity is typically lower over a prolongedperiod of time. Apart from being derived from an individual with anOXPHOS deficiency there are several methods to determine whether a cellhas an OXPHOS deficiency, such tests encompass but are not limited tooxygen consumption, ATP production capacity, and enzymatic activities ofindividual OXPHOS complexes (Chretien and Rustin J Inherit Metab Dis.2003;_26_(2-3): 189-98). It has surprisingly been found thatadministration of a compound of the invention to a cell, results inhigher amounts of OXPHOS complexes, (i.e. the mitochondria of thecells).

In the methods of the invention, the mitochondrial disorder preferablyis a disorder selected from the group consisting of Myoclonic epilepsy;Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Leber's HereditaryOptic Neuropathy (LHON); Neuropathy, Ataxia and Retinitis Pigmentosa(NARP); Mitochondrial Myopathy, Encephalopathy, Lactacidosis, Stroke(MELAS); Leigh syndrome; Leigh-like syndrome; Dominant Optic atrophy(DOA); Kearns-Sayre Syndrome (KSS); Maternally Inherited Diabetes andDeafness (MIDD); Alpers-Huttenlocher syndrome; Ataxia Neuropathyspectrum; Chronic Progressive External Ophthalmoplegia (CPEO); Pearsonsyndrome; Mitochondrial Neuro-Gastro-Intestinal Encephalopathy (MNGIE);Sengers syndrome; 3-methylglutaconic aciduria, sensorineural deafness,encephalopathy and neuro-radiological findings of Leigh-like syndrome(MEGDEL); SURF1 Leigh syndrome; myopathy; mitochondrial myopathy;cardiomyopathy; encephalomyopathy and isolated or combined oxidativephosphorylation disorders

In the methods of the invention, the condition associated withmitochondrial dysfunction preferably is a condition selected from thegroup consisting of Friedreich's Ataxia (FRDA); renal tubular acidosis;Parkinson's disease; Alzheimer's disease; amyotrophic lateral sclerosis(ALS); Huntington's disease; Barth syndrome (also known as3-Methylglutaconic aciduria type II); macula degeneration, preferablyage-related macula degeneration; developmental pervasive disorders;hearing loss, deafness; congenital muscular dystrophies,dystrophinopathies, diabetes; ageing; adverse drug effects hampering(normal) mitochondrial function, including e.g. mitochondrialdysfunction caused by nucleoside analogue reverse transcriptaseinhibitors (NRTIs), certain antibiotics and anti-epileptic drugs; andischemia and reperfusion injury, preferably ischemic reperfusion injuryafter acute myocardial infarction (AMI), after stroke, includingperinatal stroke, after haemorrhagic shock, after intestinal ischemia,after emergency coronary surgery for failed percutaneous transluminalcoronary angioplasty (PCTA), after vascular surgery with blood vesselcross clamping (e.g. of aorta, leading to skeletal muscle ischemia),after pancreatitis after manipulation of pancreatic or bile duct (ERCP),and/or after organ transplantation.

In the methods of the invention, “subject”, “individual”, or “patient”is understood to be an individual organism, preferably a vertebrate,more preferably a mammal, most preferably a human.

“Treating” a disease with the compounds and methods discussed herein isdefined as administering one or more of the compounds discussed herein,with or without additional therapeutic agents, in order to reduce oreliminate either the disease or one or more symptoms of the disease, orto retard the progression of the disease or of one or more symptoms ofthe disease, or to reduce the severity of the disease or of one or moresymptoms of the disease. “Suppression” of a disease with the compoundsand methods discussed herein is defined as administering one or more ofthe compounds discussed herein, with or without additional therapeuticagents, in order to suppress the clinical manifestation of the disease,or to suppress the manifestation of adverse symptoms of the disease. Thedistinction between treatment and suppression is that treatment occursafter adverse symptoms of the disease are manifest in a subject, whilesuppression occurs before adverse symptoms of the disease are manifestin a subject. Suppression may be partial, substantially total, or total.Because many of the mitochondrial disorders are inherited, geneticscreening can be used to identify patients at risk of the disease. Thecompounds and methods of the invention can then be administered toasymptomatic patients at risk of developing the clinical symptoms of thedisease, in order to suppress the appearance of any adverse symptoms.“Therapeutic use” of the compounds discussed herein is defined as usingone or more of the compounds discussed herein to treat or suppress adisease, as defined above. An “effective amount” of a compound is anamount of a compound which, when administered to a subject, issufficient to reduce or eliminate either one or more symptoms of adisease, or to retard the progression of one or more symptoms of adisease, or to reduce the severity of one or more symptoms of a disease,or to suppress the manifestation of a disease, or to suppress themanifestation of adverse symptoms of a disease. An effective amount canbe given in one or more administrations.

Several readily measurable clinical markers are used to assess themetabolic state of patients with mitochondrial disorders. These markerscan also be used as indicators of the efficacy of the therapy using thecompounds of the invention, as the level of a marker is moved from thepathological value to the healthy value. These clinical markers include,but are not limited to, one or more of the energy biomarkers, such aslactic acid (lactate) levels, either in whole blood, plasma,cerebrospinal fluid, or cerebral ventricular fluid; pyruvic acid(pyruvate) levels, either in whole blood, plasma, cerebrospinal fluid,or cerebral ventricular fluid; lactate/pyruvate ratios, either in wholeblood, plasma, cerebrospinal fluid, or cerebral ventricular fluid; aminoacids, in particular alanine, citrulline and proline either in wholeblood, plasma, cerebrospinal fluid, organic acids in body fluids, FGF21in serum and skeletal muscle, phosphocreatine levels, NADH (NADH+H⁺) orNADPH (NADPH+H⁺) levels; NAD or NADP levels; ATP levels; anaerobicthreshold; reduced coenzyme Q (CoQ^(red)) levels; oxidized coenzyme Q(CoQ^(ox) levels; total coenzyme Q (CoQ^(tot)) levels; oxidizedcytochrome C levels; reduced cytochrome C levels; oxidized cytochromeC/reduced cytochrome C ratio; acetoacetate levels, beta-hydroxy butyratelevels, acetoacetate/betahydroxy butyrate ratio,8-hydroxy-2′-deoxyguanosine (8-OHdG) levels; levels of reactive oxygenspecies; and levels of oxygen consumption (VO2), levels of carbondioxide output (VCO2), and respiratory quotient (VCO2/VO2).

Additional biomarkers that can be used as indicators of the efficacy ofthe therapy using the compounds of the invention include MDA, HNE,Acrolein, F₂—Isoprostanes, a decrease in GSH concentration and/orGSH/GSSG ratio, S-Glutathionylated proteins, 3-nitrotyrosine (NO₂-Tyr),3-chlorotyrosine (Cl-Tyr), o,o′-dityrosine (Di-Tyr), and Carbonylatedproteins (Dalle-Donne et al, Clinical Chem. (2006), 52(4): 601-623).

These biomarkers are particularly useful for measuring a decrease in ROSlevels (oxidative stress) after treatment with a compound of theinvention. In an embodiment of the invention, the level of one or moreof these biomarkers in a patient suffering from a disease that isassociated with an increased oxidative stress is improved to within twostandard deviations of the average level in a healthy subject. Diseasesassociated with an increased oxidative stress are outlined above, andincluding, but not limited to, Parkinson Disease, Alzheimer Disease,Epilepsy, Dementia, Asthma, Amyotrophic Lateral Sclerosis, Inbornmetabolism errors, Systemic sclerosis, Atherosclerosis, Osteoarthritis,Rheumatoid arthritis, Xenobiotics toxicity, Post-ischemic Reperfusioninjury, Hypertension, Pulmonary hypertension, Pulmonary edema, Boweldisease, Endometriosis, Diabetes, Diabetes-induced cardiac dysfunction,Diabetic nephropathy, Cancer, Embryonal rhabdomyosarcoma andAdrenoleukodystrophy.

Several of these clinical markers are measured routinely in exercisephysiology laboratories, and provide convenient assessments of themetabolic state of a subject. In one embodiment of the invention, thelevel of one or more energy biomarkers in a patient suffering from amitochondrial disease, such as Friedreich's ataxia, Leber's hereditaryoptic neuropathy, dominant optic atrophy, Leigh syndrome, SURF1, MERRF,MELAS, or KSS, is improved to within two standard deviations of theaverage level in a healthy subject after administration of an effectiveamount of a compound of the invention. In another embodiment of theinvention, the level of one or more of these energy biomarkers in apatient suffering from a mitochondrial disease, such as Friedreich'sataxia, Leber's hereditary optic neuropathy, dominant optic atrophy,Leigh syndrome, SURF1, MERRF, MELAS, or KSS is improved to within onestandard deviation of the average level in a healthy subject afteradministration of an effective amount of a compound of the invention.Exercise intolerance can also be used as an indicator of the efficacy ofa given therapy, where an improvement in exercise tolerance (i.e. adecrease in exercise intolerance) indicates efficacy of a given therapy.

Several metabolic biomarkers have already been used to evaluate efficacyof CoQ₁₀, and these metabolic biomarkers can be monitored as energybiomarkers for use in the methods of the current invention. Pyruvate, aproduct of the anaerobic metabolism of glucose, is removed by reductionto lactic acid in an anaerobic setting or by oxidative metabolism, whichis dependent on a functional mitochondrial OXPHOS. Dysfunction of theOXPHOS may lead to inadequate removal of lactate and pyruvate from thecirculation and elevated lactate/pyruvate ratios are observed inmitochondrial cytopathies (see Scriver C R, The metabolic and molecularbases of inherited disease, 7th ed., New York: McGraw-Hill, HealthProfessions Division, 1995; and Munnich et al., J. Inherit. Metab. Dis.15(4):448-55 (1992)). Blood lactate/pyruvate ratio (Chariot et al.,Arch. Pathol. Lab. Med. 118(7):695-7 (1994)) is, therefore, widely usedas a noninvasive test for detection of mitochondrial cytopathies (seeagain Scriver C R, The metabolic and molecular bases of inheriteddisease, 7th ed., New York: McGraw-Hill, Health Professions Division,1995; and Munnich et al., J. Inherit. Metab. Dis. 15(4):448-55 (1992))and toxic mitochondrial myopathies (Chariot et al., Arthritis Rheum.37(4):583-6 (1994)). Changes in the redox state of liver mitochondriacan be investigated by measuring the arterial ketone body ratio(acetoacetate/3-hydroxybutyrate:AKBR) (Ueda et al., J. Cardiol.29(2):95-102 (1997)). Urinary excretion of 8-hydroxy-2′-deoxyguanosine(8-OHdG) often has been used as a biomarker to assess the extent ofrepair of ROS-induced DNA damage in both clinical and occupationalsettings (Erhola et al., FEBS Lett. 409(2):287-91 (1997); Honda et al.,Leuk. Res. 24(6):461-8 (2000); Pilger et al., Free Radic. Res.35(3):273-80 (2000); Kim et al., Environ Health Perspect112(6):666-71(2004)).

Magnetic resonance spectroscopy (MRS) has been useful in the diagnosesof mitochondrial cytopathy by demonstrating elevations in cerebrospinalfluid (CSF) and cortical white matter lactate using proton MRS (H-MRS)(Kaufmann et al., Neurology 62(8):1297-302 (2004)). Phosphorous MRS(³¹P-MRS) has been used to demonstrate low levels of corticalphosphocreatine (PCr) (Matthews et al., Ann. Neurol. 29(4):435-8(1991)), and a delay in PCr recovery kinetics following exercise inskeletal muscle (Matthews et al., Ann. Neurol. 29(4):435-8 (1991);Barbiroli et al., J. Neurol. 242(7):472-7 (1995); Fabrizi et al., J.Neurol. Sci. 137(1):20-7 (1996)). A low skeletal muscle PCr has alsobeen confirmed in patients with mitochondrial cytopathy by directbiochemical measurements.

Exercise testing is particularly helpful as an evaluation and screeningtool in mitochondrial myopathies, and an improvement in exercisetolerance indicates the efficacy of a given therapy with a compound ofthe invention. One of the hallmark characteristics of mitochondrialmyopathies is a reduction in maximal whole body oxygen consumption(VO2max) (Taivassalo et al., Brain 126(Pt 2):413-23 (2003)). Given thatVO2max is determined by cardiac output (Qc) and peripheral oxygenextraction (arterial-venous total oxygen content) difference, somemitochondrial cytopathies affect cardiac function where delivery can bealtered; however, most mitochondrial myopathies show a characteristicdeficit in peripheral oxygen extraction (A-V 02 difference) and anenhanced oxygen delivery (hyperkinetic circulation) (Taivassalo et al.,Brain 126(Pt 2):413-23 (2003)). This can be demonstrated by a lack ofexercise induced deoxygenation of venous blood with direct AV balancemeasurements (Taivassalo et al., Ann. Neurol. 51(1):38-44 (2002)) andnon-invasively by near infrared spectroscopy (Lynch et al., Muscle Nerve25(5):664-73 (2002); van Beekvelt et al., Ann. Neurol. 46(4):667-70(1999)).

Several of these energy biomarkers are discussed in more detail asfollows. It should be emphasized that, while certain energy biomarkersare discussed and enumerated herein, the invention is not limited tomodulation, normalization or enhancement of only these enumerated energybiomarkers.

Lactic acid (lactate) levels: Mitochondrial dysfunction typicallyresults in abnormal levels of lactic acid, as pyruvate levels increaseand pyruvate is converted to lactate to maintain capacity forglycolysis. Mitochondrial dysfunction can also result in abnormal levelsof NADH+H⁺, NADPH+H⁺, NAD, or NADP, as the reduced nicotinamide adeninedinucleotides are not efficiently processed by the respiratory chain.Lactate levels can be measured by taking samples of appropriate bodilyfluids such as whole blood, plasma, or cerebrospinal fluid. Usingmagnetic resonance, lactate levels can be measured in virtually anyvolume of the body desired, such as the brain.

Measurement of cerebral lactic acidosis using magnetic resonance inMELAS patients is described in Kaufmann et al., Neurology 62(8):1297(2004). Values of the levels of lactic acid in the lateral ventricles ofthe brain are presented for two mutations resulting in MELAS, mt.3243A>Gand mt.8344A>G. Whole blood, plasma, and cerebrospinal fluid lactatelevels can be measured by commercially available equipment such as theYSI 2300 STAT Plus Glucose & Lactate Analyzer (YSI Life Sciences, Ohio).

NAD, NADP, NADH and NADPH levels: Measurement of NAD, NADP, NADH(NADH+H⁺) or NADPH (NADPH+H⁺) can be measured by a variety offluorescent, enzymatic, or electrochemical techniques, e. g., theelectrochemical assay described in US2005/0067303.

Oxygen consumption (vO₂ or VO2), carbon dioxide output (vCO₂ or VCO2),and respiratory quotient (VCO2/VO2): vO₂ is usually measured eitherwhile resting (resting vO₂) or at maximal exercise intensity (vO₂ max).Optimally, both values will be measured. However, for severely disabledpatients, measurement of vO₂ max may be impractical. Measurement of bothforms of vO₂ is readily accomplished using standard equipment from avariety of vendors, e.g. Korr Medical Technologies, Inc. (Salt LakeCity, Utah). VCO2 can also be readily measured, and the ratio of VCO2 toVO2 under the same conditions (VCO2/VO2, either resting or at maximalexercise intensity) provides the respiratory quotient (RQ).

Oxidized Cytochrome C, reduced Cytochrome C, and ratio of oxidizedCytochrome C to reduced Cytochrome C: Cytochrome C parameters, such asoxidized cytochrome C levels (Cyt C_(ox)), reduced cytochrome C levels(Cyt C^(red)), and the ratio of oxidized cytochrome C/reduced cytochromeC ratio (Cyt C^(ox))/(Cyt C^(red)), can be measured by in vivo nearinfrared spectroscopy. See, e. g., Rolfe, P., “In vivo near-infraredspectroscopy,” Annu. Rev. Biomed. Eng. 2:715-54 (2000) and Strangman etal., “Non-invasive neuroimaging using near-infrared light” Biol.Psychiatry 52:679-93 (2002).

Exercise tolerance/Exercise intolerance: Exercise intolerance is definedas “the reduced ability to perform activities that involve dynamicmovement of large skeletal muscles because of symptoms of dyspnea orfatigue” (Pina et al., Circulation 107:1210 (2003)). Exerciseintolerance is often accompanied by myoglobinuria, due to breakdown ofmuscle tissue and subsequent excretion of muscle myoglobin in the urine.Various measures of exercise intolerance can be used, such as time spentwalking or running on a treadmill before exhaustion, time spent on anexercise bicycle (stationary bicycle) before exhaustion, and the like.Treatment with the compounds or methods of the invention can result inabout a 10% or greater improvement in exercise tolerance (for example,about a 10% or greater increase in time to exhaustion, e. g. from 10minutes to 11 minutes), about a 20% or greater improvement in exercisetolerance, about a 30% or greater improvement in exercise tolerance,about a 40% or greater improvement in exercise tolerance, about a 50% orgreater improvement in exercise tolerance, about a 75% or greaterimprovement in exercise tolerance, or about a 100% or greaterimprovement in exercise tolerance. While exercise tolerance is not,strictly speaking, an energy biomarker, for the purposes of theinvention, modulation, normalization, or enhancement of energybiomarkers includes modulation, normalization, or enhancement ofexercise tolerance.

Similarly, tests for normal and abnormal values of pyruvic acid(pyruvate) levels, lactate/pyruvate ratio, ATP levels, anaerobicthreshold, reduced coenzyme Q (CoQ^(red)) levels, oxidized coenzyme Q(CoQ^(ox)) levels, total coenzyme Q (CoQ^(tot)) levels, oxidizedcytochrome C levels, reduced cytochrome C levels, oxidized cytochromeC/reduced cytochrome C ratio, acetoacetate levels, beta-hydroxy butyratelevels, acetoacetate/beta-hydroxy butyrate ratio,8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, and levels of reactiveoxygen species are known in the art and can be used to evaluate efficacyof the compounds and methods of the invention. (For the purposes of theinvention, modulation, normalization, or enhancement of energybiomarkers includes modulation, normalization, or enhancement ofanaerobic threshold).

Table 1, following, illustrates the effect that various dysfunctions canhave on biochemistry and energy biomarkers. It also indicates thephysical effect (such as a disease symptom or other effect of thedysfunction) typically associated with a given dysfunction. It should benoted that any of the energy biomarkers listed in the table, in additionto energy biomarkers enumerated elsewhere, can also be modulated,enhanced, or normalized by the compounds and methods of the invention.RQ=respiratory quotient; BMR=basal metabolic rate; HR (CO)=heart rate(cardiac output); T=body temperature (preferably measured as coretemperature); AT=anaerobic threshold; pH=blood pH (venous and/orarterial).

TABLE 1 Site of Biochemical Measurable Energy Physical dysfunction eventBiomarker Effect OXPHOS ↑ NADH Δ lactate, Metabolic Δ lactate:pyruvatedyscrasia ratio, & fatigue Δ acetoacetate:β- hydroxybutyrate ratioOXPHOS ↑ NADH Amino acid Metabolic dyscrasia & fatigue OXPHOS ↑ NADHOrganic acids Metabolic dyscrasia & fatigue OXPHOS ↑ NADH FGF21Metabolic dyscrasia & fatigue OXPHOS ↓ H⁺ gradient Δ ATP Organ dependentdysfunction OXPHOS ↓ Electron flux Δ VO₂, RQ, BMR, Metabolic ΔT, AT, pHdyscrasia & fatigue Mitochondria ↓ ATP, ↓ VO₂ Δ Work, ΔHR Exercise &cytosol (CO) intolerance Mitochondria ↓ ATP Δ PCr Exercise & cytosolintolerance Respiratory ↓ Cyt C^(Ox/Red) Δ ~ 700-900 nm Exercise Chain(NIR spectroscopy) intolerance Intermediary ↓ Catabolism Δ C¹⁴-labeledMetabolic metabolism substrates dyscrasia & fatigue Respiratory ↓Electron flux Δ Mixed venous Metabolic Chain VO₂ dyscrasia & fatigueMitochondria ↑ Oxidative Δ Tocopherol & Uncertain & cytosol stressTocotrienols, CoQ10 docosahexanoic acid Mitochondria ↑ Oxidative ΔGlutathione^(red) Uncertain & cytosol stress Mitochondria Nucleic acid Δ8-hydroxy 2- Uncertain & cytosol oxidation deoxy guanosine MitochondriaLipid Δ Isoprostane(s), Uncertain & cytosol oxidation eicasanoids CellLipid Δ Ethane Uncertain membranes oxidation (breath) Cell Lipid ΔMalondialdehyde Uncertain membranes oxidation

Treatment of a subject afflicted by a mitochondrial disease inaccordance with the methods of the invention may result in theinducement of a reduction or alleviation of symptoms in the subject,e.g. to halt the further progression of the disorder.

Partial or complete suppression of the mitochondrial disease can resultin a lessening of the severity of one or more of the symptoms that thesubject would otherwise experience. For example, partial suppression ofMELAS could result in reduction in the number of stroke-like or seizureepisodes suffered.

Any one energy biomarker or any combination of the energy biomarkersdescribed herein provides conveniently measurable benchmarks by which togauge the effectiveness of treatment or suppressive therapy.Additionally, other energy biomarkers are known to those skilled in theart and can be monitored to evaluate the efficacy of treatment orsuppressive therapy.

In a preferred embodiment, the efficacy of treatment or suppressivetherapy with the methods of the inventions can be determined using oneor more of the outcome measures of the toolbox as listed in Table 1 ofKoene et al (2013, Dev. Med. Child Neurol. 55(8): 698-706), morepreferably the efficacy is determined using one or more of the outcomemeasures of the “Common core set” in Table 1 of Koene et al (2013,supra).

The methods of the invention preferably comprise administering to asubject an effective amount of one or more compounds of the invention asherein defined above, and an acceptable carrier, excipient or vehicle,preferably a pharmaceutically or physiologically acceptable carrier,excipient or vehicle.

In yet another aspect the invention relates to the cosmetic use of thecompounds of the invention. The compounds of the invention may thus beused (in methods) to revive the skin of a treated individual,particularly in individuals with aged skin, either due to aging or dueto excessive exposure to sun. Both conditions are related to theproduction of free radicals in skin, such as ROS. By at least one ofinduction of mitochondrial filamentation, prevention or reduction ofmitochondrial fragmentation, increased expression of OXPHOS enzymes anddecreased ROS levels (e.g. superoxide levels) in a cell of saidindividual it is possible to lower the action of free radicals in theskin and at least delay further aging in the skin. As such, one can alsouse a composition of the invention as a prophylactic, i.e. to at leastreduce free radicals that would be capable to act on the skin, if leftuntreated. Thus preferably in this aspect of the invention compounds ofthe invention are applied the effect of which includes one or more ofinduction of mitochondrial filamentation, prevention or reduction ofmitochondrial fragmentation, increased expression of OXPHOS enzymes anddecreased ROS levels, preferably decreased ROS levels. Preferredcompounds having these effects are indicated herein above.

In a further aspect, the invention relates to a compound as disclosedherein for use as a conservative agent, preferably as a conservativeagent in food. It is well-known in the art that antioxidants may helpprevent the oxidation of feeds, especially fats and oils, and protectcells from free-radical damage. Therefore, food manufacturers useantioxidants as food additives to help guard against food degradationand enhance the health profile of functional foods. A compound of theinvention may function as such antioxidant and hence act as foodpreservative.

The compounds of the invention can also be used in researchapplications, such as in vitro, in vivo, or ex vivo experiments in orderto modulate one or more energy biomarkers in an experimental system.Such experimental systems can be cell samples, tissue samples, cellcomponents or mixtures of cell components, partial organs, whole organs,or organisms. Such research applications can include, but are notlimited to, use as assay reagents, elucidation of biochemical pathways,or evaluation of the effects of other agents on the metabolic state ofthe experimental system in the presence/absence of one or more compoundsof the invention.

Additionally, the compounds of the invention can be used in biochemicaltests or assays. Such tests can include incubation of one or morecompounds of the invention with a tissue or cell sample from a subjectto evaluate a subject's potential response (or the response of aspecific subset of subjects) to administration of said one or morecompounds, or to determine which compound of the invention produces theoptimum effect in a specific subject or subset of subjects. One suchtest or assay would involve 1) obtaining a cell sample or tissue samplefrom a subject or set of subjects in which modulation of one or moreenergy biomarkers can be assayed; 2) administering one or more compoundsof the invention to the cell sample(s) or tissue sample(s); and 3)determining the amount of modulation of the one or more energybiomarkers after administration of the one or more compounds, comparedto the status of the energy biomarker prior to administration of the oneor more compounds.

Another such test or assay would involve 1) obtaining a cell sample ortissue sample from a subject or set of subjects in which modulation ofone or more energy biomarkers can be assayed; 2) administering at leasttwo compounds of the invention to the cell sample(s) or tissuesample(s); 3) determining the amount of modulation of the one or moreenergy biomarkers after administration of the at least two compounds,compared to the status of the energy biomarker prior to administrationof the at least two compounds, and 4) selecting a compound for use intreatment, suppression, or modulation based on the amount of modulationdetermined in step 3).

The compounds as described herein are able to scavenge cellular reactiveoxygen species, such as superoxide. The compounds therefore may be usedin ex vivo techniques that cause an (unwanted) increase in cellularreactive oxygen species. In a further aspect, the invention thereforepertains to an ex vivo method for scavenging oxygen species in a cell,wherein the method comprises a step of exposing the cell to the compoundas defined herein.

The cell for use in a method of the invention may be any cell that canbe used in an ex vivo technique. The cell may be a prokaryotic cell oran eukaryotic cell.

In a preferred embodiment, the cell is a eukaryotic cell such as amammalian, insect, plant, fungal, yeast or algal cell. More preferably,the cell is a mammalian cell, such as e.g. a bovine, caprine, equine,ovine, porcine or primate cell, most preferably the cell is a humancell.

A preferred cell for use in the method of the invention is a somaticcell, a gamete, a gametocyte or an undifferentiated stem cell. Morepreferably, the cell is a somatic cell, and most preferably the somaticcell is a mammalian somatic cell.

The compounds of the invention scavenge cellular reactive oxygen speciessuch as superoxide and can therefore be used in ex vivo techniques tocounteract any unwanted increase in oxidative stress. Such unwantedincrease in reactive oxygen species occurs for example during thereprogramming of somatic cells into pluripotent stem cells (inducedpluripotent stem cells, iPSCs). iPSCs can subsequently be differentiatedto other cell types in vitro. Alternatively, transdifferentiation yieldsthe desired cell type without passing through a pluripotent stage. Bothtechniques require the presence of specific transcription factors tooverride the current transcriptional status and launches transcriptionalactivity characteristic of another, unrelated lineage (Novak et al, JDtsch Dermatol Ges, (2014) 12(9):789-92).

Ji et al. (supra) teach increased levels of ROS and oxidative DNA damageduring the early stages of reprogramming using the transcription factorsOCT4, SOX2, KLF4 and c-MYC. In particular, the addition of antioxidants(such as Vitamin C and N-acetyl-cysteine) reduced both ROS and genomicdouble-strand breaks. Furthermore, a significant reduction in copynumber variation (CNVs) was observed in the generated iPSCs after theaddition of antioxidants. Thus supporting the redox balance may protectthe somatic genome, leading to iPSCs with fewer genomic alterations.

In a preferred embodiment the method of the invention therefore pertainsto an ex vivo method for scavenging reactive oxygen species in a cellwherein the method comprises a step of exposing a somatic cell to acompound as defined herein and wherein the somatic cell is furtherreprogrammed to an induced pluripotent stem cell (iPSC) by exposing thecell to at least one reprogramming factor.

The somatic cell may be any somatic cell suitable for the reprogrammingthe cell into iPSC or suitable for transdifferentiating the cell intoanother cell lineage. A preferred somatic cell for use in the method ofthe invention is a fibroblast, neuronal (progenitor) cell, hepatocyte, Bcell, kidney cell, muscle cell, adrenal gland cell, keratinocyte,melanocyte, epithelial cell or a peripheral blood derived cell,preferably wherein the peripheral blood derived cell is an endothelialprogenitor cell (L-EPCs) or a cord blood derived cell type (CD34+) (Yee,J. (2010). Nature Education 3(9):25). Preferably, the somatic cell is an(embryonic) fibroblast or a peripheral blood derived cell.

Alternatively, the method of the invention relates to the use of acompound as disclosed herein for producing pluripotent stem cells,wherein the method comprises the steps of exposing a somatic cell to atleast one reprogramming factor, and exposing the somatic cell to aneffective amount of the compound of the invention.

The programming factor for use in the method of the invention may be anyfactor that induces the reprogramming of the somatic cell into an iPSCor differentiates the somatic cell into another cell lineage bytransdifferentiation. Preferably, the reprogramming factor is at leastone of Oct4, Sox2, KLF4, c-Myc, Lin28, Nanog, Glis1, Sall4, Esrrb andNr5a2.

The reprogramming of the somatic cell may require the addition of atleast 1, 2, 3, 4 or 5 reprogramming factors. These reprogramming factorsmay be added sequentially or simultaneously to the somatic cell (Liu etal, Nature Cell Biol (2013) 15(7):829-38). Particularly preferredcompositions of reprogramming factors are Oct4, Sox2, KLF4 and c-MYC(the Yamanaka factors), Oct4, Sox2, Nanog and Lin28 (the Thomsonfactors) or Sall4, Nanog, Esrrb and Lin28 (Buganim et al, Cell StemCell. (2014) 15(3): 295-309).

The compositions comprising the compounds of the invention, as describedabove, can be prepared as a medicinal or cosmetic preparation or invarious other media, such as foods for humans or animals, includingmedical foods and dietary supplements. A “medical food” is a productthat is intended for the specific dietary management of a disease orcondition for which distinctive nutritional requirements exist. By wayof example, but not limitation, medical foods may include vitamin andmineral formulations fed through a feeding tube (referred to as enteraladministration). A “dietary supplement” shall mean a product that isintended to supplement the human diet and is typically provided in theform of a pill, capsule, and tablet or like formulation. By way ofexample, but not limitation, a dietary supplement may include one ormore of the following ingredients: vitamins, minerals, herbs,botanicals; amino acids, dietary substances intended to supplement thediet by increasing total dietary intake, and concentrates, metabolites,constituents, extracts or combinations of any of the foregoing. Dietarysupplements may also be incorporated into food, including, but notlimited to, food bars, beverages, powders, cereals, cooked foods, foodadditives and candies; or other functional foods designed to promotecerebral health or to prevent or halt the progression of aneurodegenerative disease involving mitochondrial dysfunction. Ifadministered as a medicinal preparation, the composition can beadministered, either as a prophylaxis or treatment, to a patient in anyof a number of methods. The compositions may be administered alone or incombination with other pharmaceutical or cosmetic agents and can becombined with a physiologically acceptable carrier thereof. Theeffective amount and method of administration of the particularformulation can vary based on the individual subject, the condition orthe stage of disease, and other factors evident to one skilled in theart. During the course of the treatment, the concentration of thesubject compositions may be monitored to insure that the desired levelis maintained. The subject compositions may be compounded with otherphysiologically acceptable materials which can be ingested including,but not limited to, foods.

The inventions thus also pertains to pharmaceutical or cosmeticcompositions comprising one or more compounds according to theinvention. The compounds described herein can be formulated aspharmaceutical or cosmetic compositions by formulation with additivessuch as pharmaceutically or physiologically acceptable excipientscarriers, and vehicles. Suitable pharmaceutically or physiologicallyacceptable excipients, carriers and vehicles include processing agentsand drug delivery modifiers and enhancers, such as, for example, calciumphosphate, magnesium stearate, talc, monosaccharides, disaccharides,starch, gelatin, cellulose, methyl cellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-P-cyclodextrin,polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and thelike, as well as combinations of any two or more thereof. Other suitablepharmaceutically acceptable excipients are described in “Remington'sPharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991), and“Remington: The Science and Practice of Pharmacy,” Lippincott Williams &Wilkins, Philadelphia, 20th edition (2003) and 21″ edition (2005),incorporated herein by reference.

A pharmaceutical or cosmetic composition can comprise a unit doseformulation, where the unit dose is a dose sufficient to have atherapeutic or suppressive effect or an amount effective to modulate,normalize, or enhance an energy biomarker. The unit dose may besufficient as a single dose to have a therapeutic or suppressive effector an amount effective to modulate, normalize, or enhance an energybiomarker. Alternatively, the unit dose may be a dose administeredperiodically in a course of treatment or suppression of a disorder, orto modulate, normalize, or enhance an energy biomarker.

Pharmaceutical or cosmetic compositions containing the compounds of theinvention may be in any form suitable for the intended method ofadministration, including, for example, a solution, a suspension, or anemulsion. Liquid carriers are typically used in preparing solutions,suspensions, and emulsions. Liquid carriers contemplated for use in thepractice of the present invention include, for example, water, saline,pharmaceutically acceptable organic solvent(s), pharmaceuticallyacceptable oils or fats, and the like, as well as mixtures of two ormore thereof. The liquid carrier may contain other suitablepharmaceutically acceptable additives such as solubilizers, emulsifiers,nutrients, buffers, preservatives, suspending agents, thickening agents,viscosity regulators, stabilizers, and the like. Suitable organicsolvents include, for example, monohydric alcohols, such as ethanol, andpolyhydric alcohols, such as glycols. Suitable oils include, forexample, soybean oil, coconut oil, olive oil, safflower oil, cottonseedoil, and the like. For parenteral administration, the carrier can alsobe an oily ester such as ethyl oleate, isopropyl myristate, and thelike. Compositions of the present invention may also be in the form ofmicroparticles, microcapsules, liposomal encapsulates, and the like, aswell as combinations of any two or more thereof.

Time-release or controlled release delivery systems may be used, such asa diffusion controlled matrix system or an erodible system, as describedfor example in: Lee, “Diffusion-Controlled Matrix Systems”, pp. 155-198and Ron and Langer, “Erodible Systems”, pp. 199-224, in “Treatise onControlled Drug Delivery”, A. Kydonieus Ed., Marcel Dekker, Inc., NewYork 1992. The matrix may be, for example, a biodegradable material thatcan degrade spontaneously in situ and in vivo for example, by hydrolysisor enzymatic cleavage, e.g., by proteases. The delivery system may be,for example, a naturally occurring or synthetic polymer or copolymer,for example in the form of a hydrogel. Exemplary polymers with cleavablelinkages include polyesters, polyorthoesters, polyanhydrides,polysaccharides, poly(phosphoesters), polyamides, polyurethanes,poly(imidocarbonates) and poly(phosphazenes).

The compounds of the invention may be administered enterally, orally,parenterally, sublingually, by inhalation (e. g. as mists or sprays),rectally, or topically in dosage unit formulations containingconventional nontoxic pharmaceutically or physiologically acceptablecarriers, adjuvants, and vehicles as desired. For example, suitablemodes of administration include oral, subcutaneous, transdermal,transmucosal, iontophoretic, intravenous, intraarterial, intramuscular,intraperitoneal, intranasal (e. g. via nasal mucosa), subdural, rectal,gastrointestinal, and the like, and directly to a specific or affectedorgan or tissue. For delivery to the central nervous system, spinal andepidural administration, or administration to cerebral ventricles, canbe used. Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices. Theterm parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection, or infusiontechniques. The compounds are mixed with pharmaceutically acceptablecarriers, adjuvants, and vehicles appropriate for the desired route ofadministration. Oral administration is a preferred route ofadministration, and formulations suitable for oral administration arepreferred formulations. The compounds described for use herein can beadministered in solid form, in liquid form, in aerosol form, or in theform of tablets, pills, powder mixtures, capsules, granules,injectables, creams, solutions, suppositories, enemas, colonicirrigations, emulsions, dispersions, food premixes, and in othersuitable forms. The compounds can also be administered in liposomeformulations. The compounds can also be administered as prodrugs, wherethe prodrug undergoes transformation in the treated subject to a formthat is therapeutically effective. Additional methods of administrationare known in the art.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in propylene glycol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable non-irritating excipient such as cocoabutter and polyethylene glycols that are solid at room temperature butliquid at the rectal temperature and will therefore melt in the rectumand release the drug.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also compriseadditional substances other than inert diluents, e.g., lubricatingagents such as magnesium stearate. In the case of capsules, tablets, andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavouring, and perfuming agents.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidylcholines (lecithins), both natural and synthetic. Methods toform liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y., p.33 et seq (1976).

The invention also provides articles of manufacture and kits containingmaterials useful for treating, preventing, or suppressing symptomsassociated with a mitochondrial disorder or with a condition associatedwith mitochondrial dysfunction. The article of manufacture comprises acontainer with a label. Suitable containers include, for example,bottles, vials, and test tubes. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition having an active agent that is effective for treating,preventing, or suppressing symptoms associated with a mitochondrialdisorder or with a condition associated with mitochondrial dysfunction.The active agent in the composition is one or more of the compounds ofthe invention. The label on the container preferably indicates that thecomposition is used for treating, preventing, or suppressing symptomsassociated with a mitochondrial disorder or with a condition associatedwith mitochondrial dysfunction, and may also indicate directions foreither in vivo or in vitro use, such as those described above.

The invention also provides kits comprising any one or more of thecompounds of the invention. In some embodiments, the kit of theinvention comprises the container described above. In other embodiments,the kit of the invention comprises the container described above and asecond container comprising a buffer. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for performing any methods described herein.

In other aspects, the kits may be used for any of the methods describedherein, including, for example, methods for treating, preventing, orsuppressing symptoms associated with a mitochondrial disorder or with acondition associated with mitochondrial dysfunction.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost to which the active ingredient is administered and the particularmode of administration. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, body area, body mass index (BMI),general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the type,progression, and severity of the particular disease undergoing therapyor condition to be treated. The unit dosage chosen is usually fabricatedand administered to provide a defined final concentration of drug in theblood, tissues, organs, or other targeted region of the body. Theeffective amount for a given situation can be readily determined byroutine experimentation and is within the skill and judgment of theordinary clinician or skilled person.

Examples of dosages which can be used are an effective amount of thecompounds of the invention within the dosage range of about 0.1 g/kg toabout 300 mg/kg, or within about 1.0 g/kg to about 40 mg/kg body weight,or within about 1.0 g/kg to about 20 mg/kg body weight, or within about1.0 g/kg to about 10 mg/kg body weight, or within about 10.0 g/kg toabout 10 mg/kg body weight, or within about 100 μg/kg to about 10 mg/kgbody weight, or within about 1.0 mg/kg to about 10 mg/kg body weight, orwithin about 10 mg/kg to about 100 mg/kg body weight, or within about 50mg/kg to about 150 mg/kg body weight, or within about 100 mg/kg to about200 mg/kg body weight, or within about 150 mg/kg to about 250 mg/kg bodyweight, or within about 200 mg/kg to about 300 mg/kg body weight, orwithin about 250 mg/kg to about 300 mg/kg body weight. Other dosageswhich can be used are about 0.01 mg/kg body weight, about 0.1 mg/kg bodyweight, about 1 mg/kg body weight, about 10 mg/kg body weight, about 20mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg bodyweight, about 50 mg/kg body weight, about 75 mg/kg body weight, about100 mg/kg body weight, about 125 mg/kg body weight, about 150 mg/kg bodyweight, about 175 mg/kg body weight, about 200 mg/kg body weight, about225 mg/kg body weight, about 250 mg/kg body weight, about 275 mg/kg bodyweight, or about 300 mg/kg body weight. Compounds of the presentinvention may be administered in a single daily dose, or the total dailydosage may be administered in divided dosage of two, three or four timesdaily.

While the compounds of the invention can be administered as the soleactive pharmaceutical or cosmetic agent, they can also be used incombination with one or more other agents used in the treatment orsuppression of disorders. Representative agents useful in combinationwith the compounds of the invention for the treating, preventing, orsuppressing symptoms associated with a mitochondrial disorder or with acondition associated with mitochondrial dysfunction include, but are notlimited to, Coenzyme Q, vitamin E, idebenone, MitoQ, Szeto-Schillerpeptides, EPI-743, vitamin K and analogues thereof, naphtoquinones andderivatives thereof, other vitamins, and antioxidant compounds.

When additional active agents are used in combination with the compoundsof the present invention, the additional active agents may generally beemployed in therapeutic amounts as indicated in the Physicians' DeskReference (PDR) 53rd Edition (1999), which is incorporated herein byreference, or such therapeutically useful amounts as would be known toone of ordinary skill in the art. The compounds of the invention and theother therapeutically active agents can be administered at therecommended maximum clinical dosage or at lower doses. Dosage levels ofthe active compounds in the compositions of the invention may be variedso as to obtain a desired therapeutic response depending on the route ofadministration, severity of the disease and the response of the patient.When administered in combination with other therapeutic agents, thetherapeutic agents can be formulated as separate compositions that aregiven at the same time or different times, or the therapeutic agents canbe given as a single composition.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the elements is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

DESCRIPTION OF THE FIGURES

FIG. 1 . Effect of L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor ofglutathione synthesis, on the viability of primary human fibroblastsderived from healthy individuals or derived from Complex-I deficientpatients. Two days after treatment of 48 h with 200 μM BSO, cells werewashed and stained with Calcein-AM. Cell viability was determined as afunction of fluorescence intensity.

FIG. 2A-FIG. 2H Effect of the compounds on oxidative stress-induced celldeath. Primary human fibroblasts derived from Complex-I deficientpatients were treated with increasing concentrations of the compounds incombination with 200 μM BSO. After 2 days the cells were washed, stainedwith Calcein-AM and the fluorescence was measured. Cell viability isdepicted as normalized against untreated cells. Each graph depicts thepotency of the closed form and corresponding open form of the compoundto prevent cell death at selected concentrations. FIG. 2A S,R—X^(II)(R⁴═H, X═Cl) and S,R—X (R⁴═H, X=formate) in addition to the knowncompounds EPI743 and Idebenone, FIG. 2B Trolox and open Trolox, FIG. 2CR-T^(II) (R⁴═H, X═Cl) and R-T (R⁴═H, X=formate), FIG. 2D R,R—N^(II)(R⁴═H, X═Cl) and R,R—N(R⁴═H, X=formate), FIG. 2E S,R—N^(II) (R⁴═H, X═Cl)and S,R—N(R⁴═H, X=formate), FIG. 2F R,R—X^(II) (R⁴═H, X═Cl) and R,R—X(R⁴═H, X=formate), FIG. 2G R,S—X^(II) (R⁴═H, X═Cl) and R,S—X (R⁴═H,X=formate) and FIG. 2H R,trans-AE^(II) (R⁴═H, X═Cl) and R,trans-AE(R⁴═H, X=formate).

FIG. 3 . The effect of the compounds on cellular ROS levels. Primaryhuman fibroblasts derived from Complex-I deficient patients wereincubated with 5 μM CM-H₂DCFDA for 20 minutes, followed by the additionof the compound EPI743, Idebenone, S,R—X^(II) (R⁴═H, X═Cl) or S,R—X(R⁴═H, X=formate), all with a final concentration 10 μM. Untreated cells(vehicle) served as controls. Approximately 8 min. after the addition ofthe compounds, H₂O₂ was added to a final concentration of 100 μM andCM-DCF fluorescence was measured for 30 minutes.

FIG. 4A-FIG. 4E Effect of the compounds on cellular superoxideproduction. Primary human fibroblasts derived from Complex-I patientswere incubated with increasing concentrations of the compounds. Thefollowing days cells were stained with Hydroethidine (Het) (10 μM) andfluorescence was measured. Results are depicted as percentage ofvehicle-treated cells. FIG. 4A S,R—X^(II) (R⁴═H, X═Cl) and S,R—X (R⁴═H,X=formate), in addition to the known compounds EPI743 and Idebenone,FIG. 4B R-T (R⁴═H, X═Cl) and R-T (R⁴═H, X=formate), FIG. 4C R,R—X^(II)(R⁴═H, X═Cl) and R,R—X (R⁴═H, X=formate), FIG. 4D R,S—X^(II) (R⁴═H,X═Cl) and R,S—X (R⁴═H, X=formate) and FIG. 4E R,trans-AE^(II) (R⁴═H,X═Cl) and R,trans-AE (R⁴═H, X=formate).

EXAMPLES Example 1. Syntheses of the Compounds

Synthesis of the compounds according to the invention was performed byfirst preparing the closed chroman derivative of general structure (II).These closed-form derivatives of the compounds according to theinvention are designated with an superscript II. For example, the closedform of compound T as defined above is referred to as compound T^(II).Compounds of general structure (II) are prepared according to WO2014/011047.

Unless noted otherwise, materials were purchased from commercialsuppliers and used as received. CH₂Cl₂ (DCM) was freshly distilled fromcalcium hydride. All air and moisture sensitive reactions were carriedout under an inert atmosphere of dry nitrogen. Column chromatography wasperformed using Acros silica gel (0.035-0.070 mm, 6 nm).

GENERAL PROCEDURE A for the EDCI/HOAt coupling of amines to Trolox™: Toa mixture of Trolox™ (1 eq) and amine (1 eq) in DMF (dry, ˜0.2M) undernitrogen atmosphere were added EDCI.HCl (1.1 eq) and HOAt (0.1 eq). Themixture was stirred at room temperature until complete conversion(LCMS). The mixture was diluted with H₂O (20 mL) and extracted withEtOAc (3×20 mL). The combined organic phases were successively washedwith 0.5M KHSO₄ (20 mL), sat. aq. NaHCO₃ (20 mL) and brine (3×20 mL).The organic phase was dried over Na₂SO₄, filtered and concentrated invacuo, to obtain intermediate A.

GENERAL PROCEDURE B for BOC-deprotection: To a solution of intermediateA (1 eq) in DCM (˜0.03M) was added 4N HCl in dioxane (36 eq). Themixture was stirred at room temperature until complete conversion(LCMS), concentrated, coevaporated with DCM (2×), purified by reversedphase column chromatography (H₂O+0.01% (w/w) formic acid/MeCN) andfreeze-dried.

GENERAL PROCEDURE C for the ring opening of the chroman derivatives: Thecompound of formula (II) was oxidized with cerium ammonium nitrate (CAN)or with Iron(III) chloride hexahydrate in the presence of water andacetonitrile as solvent. The compound of formula (II) (0.2 mmol; 1 eq)was dissolved in MeCN (4 ml). Cerium(IV) diammonium nitrate (2.1 eq) wasdissolved in H₂O (800 μl) to form an orange solution, and added to thereaction mixture. The mixture was stirred for 30 min at room temperature(colour changed from orange to dark yellow). Then a sample was taken andanalysed by LCMS: in all instances complete and clean conversion ofstarting material into one peak with mass of the desired compound wasobserved. The reaction mixture was quenched by addition of solid NaHCO₃(3 eq.). The mixture (colour changed from dark yellow to yellow) wasstirred for 45 min at room temperature and then the MeCN was removedunder reduced pressure. 3 ml H₂O was added and the solids were filteredoff and washed with water. The filtrate was partially concentrated anddirectly purified by reversed phase column chromatography (12 g C18material, eluens MeCN and H₂O+0.01% (w/w) formic acid, collected at 225nm and 265 nm): (1) 3 min 0% MeCN; (2) 13 min 0% to 100% MeCN; and (3) 3min 100% MeCN. The pure fractions were combined and freeze-driedovernight to obtain the product in 40-80% yield as a fluffy solid.

All compounds of general structure (II) and of general structure (I) areobtained as formate salts, in other words R⁴═H, X=formate. These formatesalts are used in the examples here below. Thus, even if not indicated,R⁴═H and X=formate. Compound S,R—X was also prepared as HCl salt, byusing 0.01% (w/w) HCl solution during reversed phase columnchromatography. In examples 2-4, the HCl salt of compound S,R—X was alsotested, and no difference in activity compared to the formate salt ofcompound S,R—X, of which the results are present below, was observed.

Example 2. Effect of the Compounds on Oxidative Stress-Induced CellDeath

Methods: To assess the ability of the compounds to protect patient cellsagainst oxidative stress-induced cell death an assay was establishedusing stressed primary human fibroblasts derived from a Complex-Ideficient patient. Utilizing the inherent oxidative stress offibroblasts from patients with mitochondrial disease, their oxidativeburden was further increased by depleting cellular glutathione with aninhibitor of glutathione synthesis, L-buthionine-(S,R)-sulfoximine(BSO). As a result, while fibroblasts from healthy individuals retainedfull viability, patient fibroblasts exhibited complete cell death within48 h of the BSO insult (200 μM) (FIG. 1 ).

Cells were seeded at a density of 3000 cells/well in a 96-well formatplate and incubated with increasing concentrations of compounds incombination with BSO (200 μM, Sigma-Aldrich). Two days after treatment,the cells were washed twice and stained with a solution of 5 μMCalcein-AM (Life technologies C3100MP) during 25 min in 199 mediumwithout phenol red (Life technologies, 11043-023) at 37° C., 5% CO₂.After 2 washes with PBS the plate was read on a fluorescence platereader (Fluostar Omega, BMG labtech) and the percentage of cellviability was determined as a function of fluorescence intensity (FIG. 2).

Results: The tested compounds are shown in the table below. Except forTrolox and open Trolox, all compounds are in salt form, i.e. R⁴═H andX=formate.

Closed compounds Open compounds

For each experiment, the closed and corresponding open compound wastested for the ability to protect cells against oxidative stress-inducedcell death. As shown in FIG. 2A-2H, the open compound outperformed thecorresponding closed compound in each case. Hence, the open compoundsare more potent in protecting cells against oxidative stress-inducedcell death as compared to the corresponding closed compound.

In addition as shown in FIG. 2A, the open-form compound S,R—X is alsomore potent than the known compounds EPI743 from Edison pharmaceuticaland Idebenone from Santhera in protecting the cells against oxidativestress-induced cell death. In particular, the EC₅₀ for S,R—X was 16.71(+/−5.19) nM, while the EC₅₀ for EPI743 and Idebenone was respectively35.32 (+/−4.12) nM and 1469.70 (+/−97.10) nM.

Example 3. Effect of the Compounds on Cellular ROS Levels

Methods: CM-H₂DCFDA is a cell-permeable reporter molecule for reactiveoxygen species (ROS) that is converted into non-fluorescent andmembrane-impermeable CM-H₂DCF following removal of its acetate groups byintracellular esterases. Upon oxidation by ROS, CM-H₂DCF is convertedinto fluorescent CM-DCF. It is widely accepted that a wide variety ofROS can be responsible for the CM-H₂DCF oxidation, making it a suitablereporter of cellular oxidant levels. The average cellular CM-DCFfluorescence intensity is considered an indirect measure of cellular ROSlevels.

The effect of the compounds on the intracellular ROS levels was measuredin response to an induction of ROS by hydrogen peroxide. Primary humanfibroblasts derived from a patient with mitochondrial disease wereseeded at a density of 2500 cells/well in a 96-well format plate. Thefollowing day, the culture medium was replaced with 100 μl M199 mediumwithout FBS and phenol red and containing CM-H₂DCFDA at a finalconcentration of 5 μM (Life Technologies). The cell culture platecontaining CM-H₂DCFDA was placed for 20 minutes at 37° C., 5% CO₂. Next,the cells were washed twice with PBS, and 100 μl M199 medium without FBSand phenol red and containing the compounds (final conc. 10 μM) wasadded to each well. Wells without cells were used to correct for thebackground fluorescence. The plate was read on a fluorescence platereader (Fluostar Omega, BMG labtech) in a kinetic mode with a 2 mininterval cycle. After 4 cycles, H₂O₂ (final concentration 100 μM) wasadded using onboard injectors and the fluorescence measurement wasresumed for 1 hour. After background correction the fluorescenceintensities were plotted as a function of time.

Results: As depicted in FIG. 3 , the addition of H₂O₂ led to asignificant increase in cellular CM-DCF fluorescence. CM-DCFfluorescence intensity is a measure of cellular ROS levels, indicatingthat H₂O₂ increased intracellular ROS levels. This H₂O₂-mediatedinduction of cellular ROS levels was slightly diminished after theaddition of the commercially available compound Idebenone from Santheraor after the addition of EPI743 from Edison pharmaceutical. The additionof S,R—X^(II) further damped the H₂O₂-mediated induction of ROS levels.Strikingly the compound S,R—X, which is the corresponding open form ofS,R—X^(II), was significantly more potent than S,R—X^(II) (and EPI743and Idebenone) in limiting H₂O₂-mediated induction of ROS levels (FIG. 3).

Example 4. Effect of the Compounds on Intracellular SuperoxideProduction

Methods: HEt (Hydroethidine) is a non-fluorescent compound, which canenter the cells freely. There it is oxidized by superoxide to itsfluorescent products E⁺ and 2OHE⁺, which accumulate in negativelycharged cellular compartments (i.e. nucleus and mitochondria). E⁺+2OHE⁺fluorescence is thus considered a measure of superoxide productionwithin the cell.

The effect of the compounds on the intracellular superoxide levels wasmeasured. Primary human fibroblasts obtained from a patient withmitochondrial disease were seeded at a density of 3000 cells/well in a96-well format. The following day, the culture medium was replaced with100 μl medium containing the compounds at different concentrations.After approximately 24 hours the cells were incubated with 100 μL mediumwithout FBS and phenol red and containing HEt at a final concentration10 μM (Life Technologies). The cell culture plate containing HEt wasplaced for 10 minutes at 37° C., 5% CO₂. Next, the cells were washedtwice with medium, placed in culture medium without FBS and phenol redand visualized by fluorescence microscopy (BD Pathway 855,BDBiosciences). From the obtained images superoxide levels were analysedusing using Image Pro plus (Media Cybernetics) software.

Results: Selected compounds were tested for their superoxide scavengingcapabilities. As indicated in FIG. 4A, the compounds S,R—X^(II), EPI743(Edison pharmaceutical) and Idebenone (Santhera) demonstrated only alimited ability to scavenge superoxide. In contrast the compound S,R—X,which is the corresponding open-form of S,R—X^(II), significantlylowered superoxide levels (FIG. 4A). In fact as indicated in FIG. 4A-E,each of the tested open-form compounds was more potent than thecorresponding closed-form compound in scavenging superoxide, especiallyat higher concentrations. Hence, the open-form compounds are more potentthan their closed counterparts in scavenging intracellular superoxide.

The invention claimed is:
 1. A compound of general structure (I):

wherein wherein L is selected from —CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—NH—C(NH₂)═, —CH₂—CH₂—NH—C(O)—CH₂—NH—C(NH₂)═,—CH₂—CH₂—CH₂—NH—C(NH₂)═, —CH₂—CH₂—NH—C(Me)═,—CH₂—CH₂—NH—C(O)—CH₂—NH—C(Me)═, —CH₂—CH₂—CH₂—NH—C(Me)═,—CH₂—CH₂—NR^(1′)—C(NH₂)═, —C(CO₂H)—CH₂—CH₂—CH₂—,—C(CO₂H)—CH₂—CH₂—CH₂—NH—C(NH₂)—, —C(CO₂H)—CH₂—, —C(CO₂H)—CH₂—CH₂—,—C(CO₂H)—CH₂—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—CH₂—,—CHR^(2′)—C(O)—, —CHR^(2′)—CH₂—, —CHR⁵—CH₂—NR^(5′)—C(Me)═,—CHR^(2′)—CH₂—CH₂—, —CH₂—CH₂—CHR^(1′)—,—CH₂—CH₂—CHR^(1′)—NH—C(O)—C(Me)-, —CH₂—CHR^(1′)—,—CH₂—CHR^(1′)—NH—C(Me)═, or —CHR⁵—CH₂—CH₂—CHR^(5′)—; R¹ and R² are eachindependently selected from H, C₁-C₆ alkyl or C₁-C₆ alkenyl, or R¹ isjoined with R^(1′) in a cyclic structure and/or R² is joined with R^(2′)in a cyclic structure, or R¹ and R² are joined together and thus form asecond linker between the amide nitrogen atom and the distal nitrogenatom (N*) wherein L is a linker comprising 1 to 10 optionallysubstituted backbone atoms selected from carbon, nitrogen and oxygen; R³is selected from H, C₁-C₆ alkyl or C₁-C₆ alkenyl, wherein the alkyl oralkenyl moiety may be substituted with one or more halogen atoms or(halo)alkoxy moieties, or R³ is absent when the distal nitrogen atom ispart of an imine moiety; and R⁴ is absent; and R⁵ is joined with R^(5′)in a cyclic structure, and X is absent.
 2. The compound according toclaim 1, wherein R² is joined with a backbone atom of the linker L in asaturated cyclic structure.
 3. The compound according to claim 2,wherein R² is joined with a backbone atom of the linker L in apiperidine ring.
 4. The compound according to claim 1, whereinL=—C(CO₂H)(CH₂)₃—, R¹═R²═R³═H; L=—(CH₂)₄—, R¹═H, R²═R³=Me;L=—CHR^(2′)CH₂—R¹═R³═H, R²-R^(2′)═—(CH₂)₃—; L=—CHR⁵(CH₂)₂CHR^(5′)—,R¹═R²═R³═H, R⁵-R^(5′)═—(CH₂)₂—; or L=—CHR⁵(CH₂)₂CHR^(5′)—, R¹═R²═R³═H,and R⁵-R^(5′)═—(CH₂)₂—, which is in the S,R-configuration.
 5. Apharmaceutical or cosmetic composition comprising a compound accordingto claim 1 and a physiologically acceptable carrier.